Scaffolded maleimide clusters for multivalent peptide assembly

ABSTRACT

Disclosed are scaffolded maleimide clusters, methods of making said clusters and use of said clusters as templates for multivalent peptide assembly. Multiple maleimide functionalities were introduced onto a scaffold molecule by the reaction of a core-centered polyamines with methoxycarbonylmaleimide or with activated esters of maleimide-containing compounds. The scaffolded maleimides allow rapid, highly chemoselective, and high-yield ligation with thiolcontaining peptides under virtually neutral conditions at room temperature. The disclosed mild and highly efficient ligation method is extremely valuable for synthesizing large and complex multivalent peptides that may not be easily obtained by conventional ligation methods. These novel scaffolded maleimide clusters allow a highly chemoselective ligation with a thiolcontaining peptide under virtually neutral conditions, providing a new and efficient approach for multivalent peptide assembly. The disclosed mild and highly efficient ligation method is extremely valuable for synthesizing large and complex multivalent peptides that may not be easily obtained by conventional ligation methods. A series of multivalent peptides containing the sequence of the 36-mer HIV-1 inhibitor DP178 (T20), the T-helper epitope from tetanus toxoid (830-844), and the minimum epitope sequence of the potent HIV-neutralizing antibody 2F5 were synthesized. Carbohydrates and cholic acid were chosen as the scaffold because of their rigidity and mufti-functionality. Thus, the topology of the multivalent peptides can be controlled by the defined spatial orientation of the maleimide functionalities on the rigid scaffold core. The resulting multivalent gp41 peptides incorporating strands of DP178 on the monosaccharide and the cholic acid templates were found to be able to form three or four a-helix bundles. Moreover, the multivalent peptides containing strands of the long gp41 peptide DP178 were highly immunogenic and were able to raise high titers of peptide-specific antibodies in the absence of any additional adjuvant. Therefore, these and related multivalent peptides constructed on the maleimide clusters may be used as novel immunogens, potential inhibitors, protein mimics, artificial proteins, and powerful antigens for a broad range of biomedical applications.

CROSS-REFERENCE To RELATED APPLICATIONS

This application is filed under the provisions of 35 U.S.C. §371 andclaims the priority of International Patent Application No.PCT/US2003/019779 filed Jun. 20, 2003, which in turn claims priority ofU.S. Provisional Application No. 60/390,776 filed on Jun. 20, 2002.

BACKGROUND OF THE INVENTION Field of the Invention

The instant invention is directed towards carbohydrate-based, cholicacid-based, and other scaffolded maleimide clusters useful inmultivalent peptide and protein assembly, multivalent peptides andproteins comprising said maleimide clusters and methods of using saidmaleimide clusters and multivalent peptides and proteins.

Template assembled multivalent peptides have found wide applications inrecent years. For example, multiple antigenic peptides (MAPs), in which4-8 copies of an antigenic peptide are attached to an oligo-lysine core,were synthesized and used as synthetic vaccines against HIV and otherdiseases (Nardelli et al., 1992; Tam, 1988; Tam, 1996) and disclosed inU.S. Pat. Nos. 5,229,490 and 5,580,563. Compared to the conventionalvaccine preparations where an antigenic peptide is attached to a carrierprotein such as keyhole limpet hemocyanin (KLH) and bovine serum albumin(BSA), the MAPs constructs are chemically unambiguous andimmunologically focused, and often raise superior immune responses(Calyo-Calle et al., 1993; Nardelli et al., 1992; Tam, 1988; Tam et al.,1989; Wang et al, 1991). On the other hand, template assembledmultivalent peptides have been exploited to design artificial proteinsto study the folding and structure of proteins (Mutter et al., 1997;Tuchscherer, 1999). Enhanced inhibitory activities of peptide inhibitorswere also attained through multivalent peptide assembly (Blaskovich etal., 2000; Lin et al., 1998; Park et al., 1999; Tam et al, 2002). Inaddition to an oligo-lysine core, (Tam, 1996) other types of templatesthat were developed for multivalent peptide construction include cyclicpeptide and derivatives (Mutter et al., 1992; Tuchscherer, 1993;Tuchscherer et al., 1992; Nefzi, et al., 1995), porphyrin molecules(Akerfeldt et al., 1992; Sasaki et al., 1989), calix [4] arene core(Blaskovich et al., 2000; Lin et al., 1998; Park et al., 1999) andcarbohydrates (Brask et al., 2000; Brask et al., 2001; Jensen et al.,2000; McGeary et al., 2001; Wang, et al, 2003). Although multivalentpeptides may be assembled by a stepwise solid phase peptide synthesis onan immobilized template, the solid phase synthesis itself and thepurification of the high molecular weight multivalent peptide product toreal homogeneity, after cleavage and de-protection, present a clearchallenge (Tam, 1988). The recent development of techniques inchemoselective ligation of pre-assembled, unprotected peptide segmentshas significantly enhanced our ability to synthesize large, complexmultivalent peptides and proteins (Mutter, 1997; Tam, 1996; Tuchschereret al., 1999). Since polypeptides usually contain a batch of functionalgroups (e.g., carboxyl, amino, and hydroxyl groups, as well as aromaticside chains), the success of the ligation approach relies on a highlychemoselective reaction between a pair of mutually reactivefunctionalities that are placed on the unprotected peptide and thescaffold, respectively, which should not react crossly with any otherfunctional groups in the polypeptides. Toward this end, twochemoselective reactions are most commonly used for multivalent peptidesynthesis. One is the reaction of aminooxy or hydrazone nucleophileswith aldehydes/ketones, which results in the formation of oximes andhydrazones, respectively (Brask et al., 2000; Rose, 1994; Shao et al.,1995; Tuchscherer, 1993). The reaction is performed under acidicconditions (pH 4-5), and the oximes or hydrazones formed are chemicallystable under acidic to neutral conditions. However, this ligation isusually not applicable for peptides that are not soluble under acidicconditions such as a series of gp41 C-peptides. The other commonly usedchemoselective reaction is the thioether formation between thiol groups(usually from cysteine residues) and bromoacetyl or chloroacetylmoieties through nucleophilic substitution (Lu et al., 1991; Robey etal., 1989). However, an efficient nucleophilic substitution between athiol-chloroacetyl (or bromoacetyl) reactive pair can efficiently takeplace only under basic conditions (pH 8-9), and side reactions betweenfree amino groups in the polypeptides and the haloacetyl groups mayoccur under the reaction conditions (Robey et al., 1989). Thelimitations and disadvantages of the currently available chemoselectiveligation methods have prompted the invention of highly efficient,generally applicable methods for multivalent peptide assembling. Thisinstant invention discloses the preparation of structurally definedscaffolded maleimide clusters and their application for the constructionof various multivalent peptides for a range of applications.

One area for the use of multivalent peptides is HIV vaccines and viralmembrane fusion inhibitors. The HIV-1 gp41 is an envelope glycoproteinthat mediates the fusion of viral and cellular membranes, a criticalstep for HIV entry and infection. It was reported that synthetic gp41C-peptides (peptides corresponding to the C-terminal ectodomain of gp41)such as T20 and C34, potently inhibit membrane fusion by bothlaboratory-adapted strains and primary isolates of HIV-1 (Malashkevichet al., 1998; Wild et al., 1993; Wild et al., 1994). The critical rolesof certain peptide domains of gp41 in mediating membrane fusion provideideal targets for developing therapeutic and preventative agents againstHIV/AIDS (Chan et al., 1998). The invention allowed the construction ofvarious multivalent gp41 peptides as mimics of the fusion-active statesof oligomeric gp41 expressed on HIV-1, which may be used as effectiveHIV-1 vaccine and inhibitors.

Another area for the use of the multivalent peptides constructed fromthe maleimide clusters is for artificial protein design. The maleimideclusters assembled on a rigid scaffold molecule such as monosaccharidesand cholic acid can well control the topology of the peptide strandsligated onto the templates, leading to the formation of secondarystructure such as α-helix bundles.

BRIEF SUMMARY OF THE INVENTION

Disclosed are carbohydrate-centered maleimide clusters, cholicacid-based maleimide clusters, and other related maleimide clusters andthe facile synthesis thereof. These maleimide clusters take advantage ofthe well-established, highly efficient Michael-type addition of a thiolgroup to a maleimide moiety (Kitagawa et al., 1976; Peeters et al.,1989). The instant scaffolded maleimide clusters allow a highlychemoselective ligation with a thiol-containing peptide under virtuallyneutral conditions, providing a new and efficient approach formultivalent peptide assembly. The disclosed mild and highly efficientligation method is extremely advantageous for synthesizing large andcomplex multivalent peptides that may not be easily obtained byconventional ligation methods. A series of multivalent peptidescontaining the sequence of the 36-mer HIV-1 inhibitor DP178 (T20), theT-helper epitope from tetanus toxoid (830-844), and the minimum epitopesequence of the potent HIV-neutralizing antibody 2F5 were synthesized.

We chose carbohydrates and cholic acid as the scaffold because the rigidring structure and the distinct configurations of multiplefunctionalities in the scaffold make them unique platforms fortopological accommodations of peptide chains. Thus, the topology of themultivalent peptides can be controlled by the defined spatialorientation of the maleimide functionalities on the rigid scaffold core.The resulting multivalent gp41 peptides incorporating strands of DP178on the monosaccharide and the cholic acid templates were found to beable to form three or four α-helix bundles. Moreover, the multivalentpeptides containing strands of the long gp41 peptide DP178 were highlyimmunogenic and were able to raise high titers of peptide-specificantibodies, in the absence of any additional adjuvant. Therefore, theseand related multivalent peptides constructed on the maleimide clustersmay be used as novel immunogens, potential inhibitors, protein mimics,artificial proteins, and powerful antigens for a broad range ofbiomedical applications.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1. Synthesis of a galactoside-based, tetravalent maleimide cluster,(a) allyl bromide, NaH/DMF, 0° C.-room temp., 94%; (b) 9-BBN, THF,reflux, then 3 M aqueous. NaOH, 30% H₂O₂, 0° C.-room temp., 84%; (c)PPh₃/I₂, DMF, 80° C., 70%; (d) NaN₃, DMF, room temp. 78%; (e) Pd/C, H₂,MeOH, room temp., 100%; (f) methoxycarbonylmaleimide, 1 M aqueousNaHCO₃, MeCN, room temp., 76%.

FIG. 2. Synthesis of a tetravalent maleimnide cluster with spacersbetween the carbohydrate core and the maleimide. (a) cysteaminehydrochloride, MeOH, hv, room temp., 80%; (b) methoxycarbonylmaleimide,1 M aqueous NaHCO₃, MeCN, room temp., 71%; (c) 6-maleimidohexanoic acidN-succinimidyl ester, 1 M aqueous NaHCO₃, THF, 0° C.-room temp., 43%.

FIG. 3. Synthesis of a β-glucopyranoside-based, pentavalent maleimidecluster with spacers between the carbohydrate core and the maleimide.(a) cysteamine hydrochloride, MeOH, hv, room temp., 80%; (b)6-maleimidohexanoic acid N-hydroxylsuccinimide ester, 1 M aqueousNaHCO₃, THF, 0° C.-room temp., 39%.

FIG. 4. Synthesis of a tetravalent maleimide cluster based uponpentaerythritol. (a) NaH/DMF, allyl bromide, 0° C.-room temp., 91%; (b)cysteamine, AIBN, MeOH, UV (254 nm), room temp., 84%; (c)Methoxycarbonylmaleimide, 1M aqueous NaHCO₃, MeCN, room temp., 50%.

FIG. 5. Synthesis of a β-cyclodextrin-centered, dendritic clustercomprising 21 maleimide functionalities. (a) NaH/DMF, allyl bromide, 0°C.-room temp., 91%; (b) cysteamnine, AIBN, MeOH, UV (254 nm), roomtemp., 68%; (c) N-hydroxylsuccinimide ester of 6-maleimidohexanoic acid,1M aqueous NaHCO₃, THF, 0° C.-room temp., 51%.

FIG. 6. Ligation of a tetravalent maleimide cluster with peptides of thesequence Ac-ELDKWAC (SEQ ID NO: 1) or Ac-CELDKWA (SEQ ID NO: 2). (a)phosphate buffer (50 mM, pH 6.5-7.5), room temp., Yields: 91% for 22;88% for 23.

FIG. 7. Ligation of peptides of the sequenceAc-YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFC (SEQ ID NO: 3) with thecarbohydrate-centered tetravalent maleimide clusters. (a) 1:1MeCN-phosphate buffer (pH 7.0), room temp., Yields: 82% for 25; 84% for26.

FIG. 8. General structures of cholic acid-based maleimide clusters.

FIG. 9. Synthesis of typical cholic acid-based maleimide clusters. (a)LiAlH4, THF, 0° C. to rt, 98%; (b) Trityl chloride, Et3N, DBU, DMF, rt,93%; (c) Allyl iodide, NaH, THF, rt to 70° C., 96%; (d)p-Toluenesulfonic acid, DCM, MeOH, 100%; (e) 2-aminoethanethiolhydrochloride, ABIN, MeOH, UV (254 nm), rt, 96%; (f) 6-maleimidohexanoicacid N-hydroxylsuccinimide ester, DCM, rt 76%; (g)N-methoxycarbonylmaleimide, Et3N, DMF, rt 78%, (h) Bromoaceticanhydride, DCM, rt, 89%; or Bromoacetic anhydride, MeCN/NaHCO₃ (pH 8.5,50:50), rt, 96%.

FIG. 10. Ligation of peptides to the cholic acid-based maleimideclusters and the bromoacetyl derivatives. (a) phosphate buffer (pH6.6)/MeCN (50:50), rt; (b) borate buffer (pH 8.5)/MeCN (50:50), rt.

FIG. 11. CD spectra of the MVP-1 and DP178. The spectra were recordedwith 10 uM of DP178 and 2.5 uM of MVP-1 in a phosphate buffer (50 mM, pH7.4) at 23° C.

FIG. 12. Antibody titers of the sera from MVP-1-immunized mice againstimmobilized DP178 at 1 month. BALB/c mice (5 per group) were injectedintraperitoneally (i.P.) at days 0, 7, 14, and 21 with 10 ug ofimmunogens without any adjuvant. The sera were collected 10 days afterthe last boost and used for the ELISAs.

FIG. 13. Antibody titers of the sera from MVP-1-immunized mice againstimmobilized DP178 at 8 month. BALB/c mice (5 per group) were boosted atmonth-8 and sera were collected 10 days after the last boost and usedfor the ELISAs.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, a multivalent peptide is a compound comprising more thanone peptide covalently attached to a scaffold compound. A peptidecomprises two or more amino acids covalently linked by a peptide bond.Any peptide that contains a cysteine residue is able to react with amaleimide moiety and form a covalent bond. A scaffold compound isdefined herein as a core compound comprising two or more reactive groupswhereby a peptide can be covalently attached. For the instant invention,scaffolds can be, but are not limited to monosaccharides, polyols andoligosaccharides. Monosaccharides that can serve as a scaffold of theinstant invention include but are not limited to dihydroxyacetone, R andL enantiomeric and anomeric forms of glyceraldehyde, threose, erythrose,erythrulose, ribose, arabinose, xylose, lyxose, ribulose, xylulolse,allose, altrose, glucose, mannose, gulose, idose, galactose, talose,psicose, fructose, sorbose and tagatose. Polyols or polyalchohols thatcan serve as a scaffold compound include, but are not limited to,glyceritol, threitol, erythritol, ribitol, arabinitol, xylitol, lyxitol,allitol, altritol, glucitol, mannitol, galactitol, talitol, gulitol,iditol, sorbitol, mannitol, glycerol, inositol, maltitol, lactitol,dulcitol and adonitol. Oligosaccharides that can serve as a scaffoldcompound include, but are not limited to, disaccharides comprising anycombination of monosaccharides described supra and cyclicoligosaccharides comprising the monosaccharides described supra.Cyclodextrin and cyclofructin are examples of cyclic oligosaccharidesthat can be used in the scaffold of the instant invention. Cyclodextrinsare cyclic (α-1, 4)-linked oligosaccharides and include, but are notlimited to 5-13 α-D-gluco-pyranose, cyclomannin, cycloaltrin andcyclogalactin. Cyclodextrins comprise a hydrophobic core, capable ofcarrying compounds. A maleimide cluster may further comprise severallinked core compounds comprising reactive maleimide moieties. A coremolecule for the instant invention also includes, but is not limited to,cholic acid, cholesterol, cyclic peptides, porphyrins and calyx [4]arene, carbohydrates and polyamines. Polyamines include, but are notlimited to, bis aminopropyl piperazine, iminobis propylamnine,methylimino bis propylamine, diamine propane, biamino butane,putrescine, cadaverine, spermidine and polyamines produced by theallylation and subsequent photoaddition of oligosaccharides. Essentiallyany compound containing a reactive group whereby a maleimide linker armcan be attached is a suitable core molecule for the instant invention.

For the scaffold of the instant invention, the reactive groups aremaleimides that form covalent bonds with peptides containing a reactivethiol group, such as a cysteine residue. As used herein, a maleimidecluster is defined as a scaffold comprising more than one maleimide.

The reactive groups of a scaffold are attached either directly to a corecompound or via linkers. The linkers are of various lengths and compriseany combination of C, N, O, P and S atoms as the backbone of the linker.These linkers between the core compound and the reactive group may be1-50 atoms in length. Such atoms include, but are not limited to,carbon, nitrogen, oxygen, phosphorous and sulfur. The linkers arepreferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 atoms inlength.

As defined herein, multivalent peptides are a maleimide cluster withcovalently attached peptides. The peptides of a multivalent peptide areeither the same or different. The instant disclosure further describescompositions comprising multivalent peptides and maleimide clusters. Acomposition may comprise multivalent peptides where the peptides areidentical, not identical or a combination of identical and non-identicalpeptides. Additionally, the scaffolded clusters of the instant inventionmay comprise not only maleimide but also maleimide and otherthiol-reactive compounds.

Pharmaceutical compositions include multivalent peptides and/ormaleimide clusters with a pharmaceutically acceptable carriers.Pharmaceutically acceptable carriers includes those approved for use inanimals and humans and include diluents, adjuvants, excipients or anyvehicle with which a compound, such as multivalent peptides and/ormaleimide clusters, is administered. Pharmaceutically acceptablecarriers include but are not limited to water, Ringer's solution,isotonic saline, oils, dextrose solutions, glycerol solutions,excipients such as starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, powdered non-fat milk, propylene glycol andethanol. Pharmaceutical compositions may also include wetting oremulsifying agents, or pH buffering compounds. Wetting or emulsifyingagents include, but are not limited to, sodium dodecyl sulfate,polyoxyethylene derivatives of fatty acids, partial esters of sorbitolanhydrides, TWEEN 80, TWEEN 20, POLYSORBATE 80, TRITON X 100, bile saltssuch as sodium deoxycholate, zwitterionic detergents such asN-dodecyl-N,N-dimethyl-2-ammonio-1 ethane sulphonate and its congenersor non-ionic detergents such as octyl-beta-D-glucopyranoside.

The multivalent peptides can be formulated into a composition in aneutral or salt form. Pharmaceutically acceptable salts, include theacid addition salts (formed with the free amino groups of the peptide)and which are formed with inorganic acids include, but are not limitedto, hydrochloric or phosphoric acids, or such organic acids as acetic,oxalic, tartaric and mandelic. Salts formed with the free carboxylgroups can also be derived from inorganic bases which include, but arenot limited to, sodium, potassium, ammonium, calcium, or ferrichydroxides, and such organic bases as isopropylamine, trimethylamine,histidine and procaine.

Adjuvants include but are not limited to alum, mineral oil, choleratoxin b-subunit, dehydroepiandrosterone sulfate, Freund's (complete andincomplete), lysolecithin, pluronic polyols, keyhole limpet hemocyanin,dinitrophenol, Bacillus Calmette-Guerin and Corynebacterium parvum.

Pharmaceutically acceptable buffers are known in the art and include butare not limited to sodium phosphate, sodium citrate, sodium acetate,TRIS glycine, HEPES, MOPS or Bis-Tris.

The multivalent peptides of the instant invention are useful in manydifferent ways. A multivalent peptide allows the delivery of peptidesfor vaccination. Compaction of DNA by polycations in conjunction withthe multivalent peptide allows delivery of DNA into cells. Suchmultivalent peptides may also comprise peptides which would guide theimport and localization into cells, and can be used to deliver drugs inaddition to DNA. Protein folding can be studied using the maleimidecluster scaffold. Peptides attached to the maleimide cluster scaffoldexhibit an increased percentage of peptides having helical structure,allowing the study of protein folding. Additionally, increasedantigenicity is obtained for the multivalent peptides, possibly due toan increased percentage of the attached peptides having an orderedfolded structure.

Enzymes may be immobilized on the maleimide cluster. Such immobilizedenzymes may provide for improved stability of the enzymes. Enzymes of apathway may be placed in proximity to each other using a maleimidecluster, improving overall activity of the enzymatic pathway by reducingsubstrate diffusion and providing for channeling of the substratebetween the enzymes.

Any compound containing a thiol group can be attached to the maleimideclusters of the instant invention. For peptides and proteins that do nothave an available reactive thiol, various methods of adding cysteine toany amino acid sequence are well known in the art. For non-peptide andnon-protein compounds, various methods of thiol addition to non-peptideand non-protein compounds are also well known in the art.

Attachment of peptides and proteins to the scaffolded maleimide clustersof the instant invention increases valency of the peptide(s) orprotein(s). With the attachment of the peptide or protein to thescaffold, the stability of the peptide or protein is increased. Suchstability is observed in native or more native conformation of thepeptide or protein when attached to the scaffold of the instantinvention. Furthermore, with the multivalency of the scaffold, adjuvantsmay be attached to the same scaffold along with antigens.

In addition to maleimide, other possible thiol reactive compounds areiodoacetic acid, bromoacetic acid, iodoacetamide and pyridyl disulfide.The disulfide linkages formed with pyridyl disulfide are cleavable bymethods well known in the art. Any number of thiol reactive groups maybe added to a core molecule or core molecules, including but not limitedto 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 41, 43, 44, 45, 46, 47, 48, 49 and 50 thiol reactivegroups.

Furthermore, multivalent peptides comprising a maleimide cluster can beused either orally or parenterally, wherein parentally as used hereinrefers to modes of administration which include intravenous,intramuscular, intraperitoneal, instrasternal, subcutaneous andintraarticular injection and infusion.

Suitable proteins for attachment to the scaffolded maleimide cluster ofthe instant invention include, but are not limited to, toleragens,vaccine peptides and proteins, receptors and mimics, ligands and mimics,toxins, transition-states analogues of multiple protein-proteininteractions, T-helper cell and B-cell epitopes, and CTL epitopes.Suitable proteins include, but are not limited to CD4, CD5, CD8, CCR5,CCR4, CCR3, CCR1, HIV-I Tat, gp41, gp120, MHC I and MHC II proteins,IL-1, IL-2, IL-4, IL-10.

A preferred embodiment of the instant invention is a maleimide clustercomprising a core molecule wherein five or more maleimides are eachattached to the core. Another preferred embodiment of the invention is amaleimide cluster comprising a carbohydrate core wherein two or moremaleimides are each attached to the core. Yet another preferredembodiment of the invention is a maleimide cluster comprising a coremolecule wherein five or more maleimides are each attached to the coreby a linker. Still another preferred embodiment of the invention is amaleimide cluster comprising a carbohydrate core wherein one, two,three, four, five or more maleimides are each attached to the core by alinker.

A preferred embodiment of the invention is a maleimide clustercomprising a cholic acid core wherein one, two, three, four, five ormore maleimides are each attached to the core. Another preferredembodiment of the invention is a maleimide cluster comprising a cholicacid core wherein one, two, three, four, five or more maleimides areeach attached to the core by a linker.

A further preferred embodiment of the invention is a maleimide clustercomprising a core molecule, wherein six or more maleimides are eachattached to the core. Yet another preferred embodiment of the inventionis a maleimide cluster comprising a core molecule, wherein seven or moremaleimides are each attached to the core.

A preferred embodiment of the invention is a maleimide clustercomprising a core molecule wherein two or more maleimides are eachattached to the core and wherein the core is selected from the groupconsisting of monosaccharides, polyols, oligosaccharides, cyclicoligosaccharides, polyamines, cholic acid, cholesterol, cyclic peptides,porphyrins and calyx [4] arene. A further preferred embodiment of theinvention is a maleimide cluster comprising a core molecule wherein twoor more maleimides are each attached to the core and wherein the core isa monosaccharide. Another further preferred embodiment of the inventionis a maleimide cluster comprising a core molecule wherein two or moremaleimides are each attached to the core and wherein the core is apolyol. Still another further preferred embodiment of the invention is amaleimide cluster comprising a core molecule wherein two or moremaleimides are each attached to the core and wherein the core is anoligosaccharide. Yet another further preferred embodiment of theinvention is a maleimide cluster comprising a core molecule wherein twoor more maleimides are each attached to the core and wherein the core isa cyclic oligosaccharide. Another further preferred embodiment of theinvention is a maleimide cluster comprising a core molecule wherein twoor more maleimides are each attached to the core and wherein the core isa cholic acid.

A preferred embodiment of the invention is a maleimide clustercomprising cyclodextrin wherein one or more maleimides are each attachedto the cyclodextrin by a linker. A preferred embodiment of the inventionis a maleimide cluster comprising at least two cores wherein each corecontains one or more maleimides. Another preferred embodiment of theinvention is a maleimide cluster comprising a polyol core, wherein oneor more maleimides are each attached to the core. A further preferredembodiment of the invention is a maleimide cluster comprising a polyolcore, wherein two or more maleimides are each attached to the core by alinker.

A further preferred embodiment of the invention is a multivalent peptideor protein comprising said maleimide clusters described supra withpeptides or proteins covalently attached to the maleimide. Anotherfurther preferred embodiment is a multivalent peptide or proteincomprising said maleimide clusters described supra with peptides orproteins covalently attached to the maleimide, wherein the covalentlyattached peptides or proteins are identical in their amino acidsequence. Still another preferred embodiment is a multivalent peptide orprotein comprising said maleimide clusters described supra with peptidesor proteins covalently attached to the maleimide, wherein the covalentlyattached peptides or proteins differ in their amino acid sequence andthere are two or more different peptides or proteins.

A preferred embodiment of the invention is a method of vaccinationcomprising administering a multivalent peptide or protein in an amountsufficient to elicit a protective immune response in an animal, whereinthe multivalent peptide or protein comprises peptides or proteinscovalently attached to said maleimide clusters, described supra. Afurther preferred embodiment of the invention is a method of vaccinationcomprising administering a multivalent peptide or protein in an amountsufficient to elicit a protective immune response in an animal, whereinthe multivalent peptide or protein comprises peptides or proteinscovalently attached to said maleimide clusters, described supra, whereinthe covalently attached peptides or proteins are identical in theiramino acid sequence. Another further preferred embodiment of theinvention is a method of vaccination comprising administering amultivalent peptide or protein in an amount sufficient to elicit aprotective immune response in an animal, wherein the multivalent peptideor protein comprises peptides or proteins covalently attached to saidmaleimide clusters, described supra, wherein the covalently attachedpeptides or proteins differ in their amino acid sequence and there aretwo or more different peptides or proteins.

A method of delivering a peptide drug comprising administering amultivalent peptide or protein containing a therapeutically effectiveamount of the peptide or protein drug to a patient in need thereof,wherein the multivalent peptide or protein comprises peptides orproteins covalently attached to said maleimide cluster s describedsupra. Another preferred embodiment is a method of delivering a peptidedrug comprising administering a multivalent peptide or proteincontaining a therapeutically effective amount of the peptide or proteindrug to a patient in need thereof, wherein the multivalent peptide orprotein comprises peptides or proteins covalently attached to saidmaleimide clusters, described supra, wherein the covalently attachedpeptides or proteins are identical in their amino acid sequence. Stillanother preferred embodiment of the invention is a method of deliveringa peptide drug comprising administering a multivalent peptide or proteincontaining a therapeutically effective amount of the peptide or proteindrug to a patient in need thereof, wherein the multivalent peptide orprotein comprises peptides or proteins covalently attached to saidmaleimide clusters, described supra, wherein the covalently attachedpeptides or proteins differ in their amino acid sequence and there aretwo or more different peptides or proteins.

A preferred embodiment of the invention is a method of making amultivalent peptide or protein comprising contacting peptides orproteins containing a thiol group with said maleimide cluster, describedsupra. A further preferred embodiment of the invention is a method ofmaking a multivalent peptide or protein comprising contacting peptidesor proteins containing a thiol group with said maleimide cluster,described supra, wherein the peptides or proteins are identical in aminoacid sequence. Another further preferred embodiment of the invention isa method of making a multivalent peptide or protein comprisingcontacting peptides or proteins containing a thiol group with saidmaleimide cluster, described supra, wherein the peptides or proteinsdiffer in their amino acid sequence and there are two or more differentpeptides or proteins. Still another further preferred embodiment of theinvention is a method of making a multivalent peptide or proteincomprising contacting peptides or proteins containing a thiol group withsaid maleimide cluster, described supra, wherein the peptides orproteins are identical in amino acid sequence.

A preferred embodiment of the invention is a method of producingpolyclonal antibodies to a peptide or protein comprising administeringsaid peptide or protein covalently attached to said maleimide clusters,described supra, to an animal and isolating the polyclonal antibodiesproduced.

A preferred embodiment of the invention is a method of producingmonoclonal antibodies to a peptide or protein comprising administeringsaid peptide or protein covalently attached to said maleimide clusters,described supra, to an animal, isolating the spleen to producehybridomas and isolating the monoclonal antibodies produced.

A preferred embodiment of the invention is a method for targetingspecific cells or cellular organelles for delivery of a compoundcomprising said maleimide clusters, discussed supra, comprising anattached targeting protein.

A preferred embodiment of the invention is a method of making amultivalent peptide or protein comprising adding a cysteine to saidpeptide or protein and contacting with said maleimide clusters,discussed supra, to form a covalent bond. A preferred embodiment of theinvention is a method of making a multivalent compound comprisingcovalently adding a thiol to a compound and contacting with saidmaleimide clusters, described supra, to form a covalent bond.

A preferred embodiment of the invention is a maleimide clustercomprising at least two cores wherein each core contains one, two,three, four, five or more maleimides. A preferred embodiment of theinvention is a multivalent peptide comprising the maleimide clustercomprising a carbohydrate core or a cholic acid core or any other coreswherein three or more maleimides are each attached to the core by alinker with peptides covalently attached to the maleimides. A preferredembodiment of the invention is a multivalent peptide comprising amaleimide cluster comprising at least three cores wherein each corecontains three or more maleimides with peptides covalently attached tothe maleimides. Another preferred embodiment is a linker of themaleimide cluster comprising C, N, O, P or S atoms. A more preferredembodiment is where the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49 or 50 atoms in length and comprising any combination of C, N, O, P orS atoms.

EXAMPLE 1

A galactoside-based, tetravalent maleimide cluster (7) was synthesizedthrough a series of efficient chemical transformations (FIG. 1). First,compound (1) was allylated with allyl bromide to give the tetra-O-allylgalactoside (2)(94%). The tetra-allyl derivative was then subject toregioselective hydroboration with 9-borabicyclo-[3.3.1]nonane (9-BBN),and subsequent alkaline oxidation with H₂O₂ to give thetetra-O-(3-hydroxypropyl) galactoside (3) in 84% yield. To synthesizethe tetra-amino derivative (6) that is required for introducingmaleimide groups, the tetraol (3) was reacted withtriphenylphosphine-iodine in DMF to give the tetraiodide (4)(70%), whichwas then converted into the azido-compound (5) by treatment with NaN₃ inDMF. Catalytic hydrogenation of 5 afforded the tetra-amino derivative(6) in a quantitative yield. Finally, simultaneous introduction of 4maleimide groups was achieved by treating the amine (6) withmethoxycarbonylmaleimide in aqueous MeCN containing NaHCO₃ to give thetetravalent maleimide cluster (7) in 76% yield (FIG. 1).

EXAMPLE 2

The synthesis of maleimide clusters with variable length of spacersbetween the carbohydrate core and the maleimide was easily achievable byextending the spacers during the synthesis. The length of spacersbetween the carbohydrate core and the peptide chains is an importantfactor to determine the orientation and intra-molecular interaction ofthe peptide chains, which will eventually affect the properties of theresulting multivalent peptides (Peczuh et al., 2000; Tam, 1996) For thepurpose, two tetravalent maleimide clusters (compounds 9 and 10) thathave longer spacers between the maleimide and the carbohydrate core weresynthesized (FIG. 2). Briefly, four amino functionalities wereintroduced into the tetra-O-allyl derivative (2) by photoaddition withcysteamine in MeOH (Dubber et al., 1998). Instead of using a largeexcess of cysteamine hydrochloride as previously reported (Dubber etal., 1998), we used only 3 molar equivalent per OH of cysteaminehydrochloride and monitored the reaction by measuring ¹H-NMR. When thereaction is proceeding, the signals at δ5.10-6.05 (for the allyl groups)decrease and the new signals at δ2.68-2.90 (for SCH₂) increase. Afterdisappearance of the allyl signals, the resulting product (8) wasreadily isolated in 80% yield by SEPHADEX G-15 gel filtrationchromatography. We found that using less cysteamine hydrochloride didnot affect the efficiency of the reaction but greatly facilitated thepurification of the product by gel filtration. Treatment of 8 withmethoxycarbonylmaleimide gave the tetravalent maleimide cluster (9) in71% yield after chromatographic purification. On the other hand,coupling amine (8) with the N-hydroxylsuccinimide ester of6-maleimidohexanoic acid afforded the tetravalent maleimide cluster (10)in 43% yield (FIG. 2).

EXAMPLE 3

The established synthetic schemes are equally useful for the synthesisof maleimide clusters on different carbohydrate cores, which will allowthe presentations of peptide chains in distinct orientations as well asin different valencies. As an example, a β-glucopyranoside-based,pentavalent maleimide cluster was readily synthesized (FIG. 3). Briefly,the penta-O-allyl β-glucoside (11), which was prepared according to thereported procedure (Leydet et al., 1997), was converted into theamino-compound (12) in 80% yield by photoaddition with cysteamine.Compound (12) was then reacted with the N-hydroxylsuccinimide ester of6-maleimidohexanoic acid, giving the penta-valent maleimide cluster (13)in 39% yield (FIG. 3).

EXAMPLE 4

The scaffold is not limited to sugar molecules. Other organic moleculescan also be used as the core to prepare maleimide clusters throughappropriate chemical modifications. As shown in FIG. 4, pentaerythritol(14) was efficiently converted into a tetravalent maleimide cluster (17)by three chemical steps in high yields. In contrast tocarbohydrate-centered maleimide clusters that present the maleimidefunctionalities in an asymmetric three-dimensional fashion, compound 17presents the four maleimide functionalities in a non-discriminatingspatial arrangement. Both types of presentations will be useful fordifferent purposes.

EXAMPLE 5

A special class of oligosaccharides, the cyclodextrins, was exploited asthe scaffold for the construction of multivalent maleimide clusters.FIG. 5 showed the synthesis of β-cyclodextrin-centered, dendriticcluster that has 21 maleimide functionalities arranged along the ridgeof the two faces of the cyclodextrin molecules. Briefly, multiple aminofunctionalities were introduced into β-cyclodextrin by our establishedprocedures (allylation and subsequent photoaddition) to give thepolyamine 20 (Ni et al., 2002). This polyamine was then reacted with theN-hydroxylsuccinimide ester of 6-maleimido-hexanoic acid to provide thecyclodextrin-centered maleimide cluster (21)(FIG. 5). This is thebiggest maleimide cluster so far described that has a well-definedchemical structure.

EXAMPLE 6

To examine the usefulness of the synthetic maleimide clusters formultivalent peptide assembling, we first set to prepare two multipleantigenic peptides, using 2F5's epitope ELDKWA (SEQ ID NO: 4) as themodel peptides. 2F5 is one of the few broadly neutralizing antibodiesisolated that can neutralize various primary HIV-1 strains. Theneutralizing epitope of 2F5 was mapped to be ELDKWA (SEQ ID NO:4)(Muster et al., 1993) For the coupling, a cysteine residue wasintroduced into the epitope sequence either at the C-terminus(peptide 1) or the N-terminus (peptide 2) during the solid phase peptidesynthesis. As expected, the ligation between peptide 1 and the malemiidescaffold 10 is extremely fast and efficient at neutral pH at ambienttemperature (FIG. 6). A simple HPLC purification gave the tetravalentpeptide 22 in 91% yield. We also found that the ligation reaction isequally efficient between pH 6.5-7.5 in an aqueous buffer, which isparticularly useful for different peptides that may behave differentlyunder distinct pH. Similarly, the coupling of 10 with the peptide2 thathas the cysteine residue at the N-terminus gave the tetravalent 23 inhigh yield (FIG. 6).

EXAMPLE 7

The maleimide clusters are equally applicable for larger and morecomplex peptides. Accordingly, the tetravalent gp41 peptides 25 and 26,each of which contains 4 strands of the 36-mer HIV-1 gp41 peptide DP178(T20) were synthesized (FIG. 7). The peptide exhibits potent and broadlyinhibitory activities against different strains of HIV through blockingviral membrane fusion (Wild et al., 1993; Wild et al., 1994), and iscurrently in clinical trials for the treatment of AIDS (Kilby et al.,1998). In order to ligate the peptide to the maleimide cluster, acysteine residue was introduced at the C-terminus of the peptide duringsolid phase peptide synthesis. The cysteine-containing peptide (24) wasthus synthesized using the Fmoc chemistry and purified by reverse phaseHPLC. While peptide (24) is insoluble in aqueous buffers below pH 6.5,it is readily soluble under neutral to slightly alkaline conditions (pH7.0-7.5). Interestingly, the initial attempt to ligate the peptide (24)with the maleimide cluster (7) in a phosphate buffer (50 mM, pH 7.2)failed to give the desired tetravalent peptide. The reaction resulted ina polymer-like solid that, after lyophilization, can hardly dissolveagain in aqueous buffers or aqueous organic solvents, or in pure organicsolvents such as DMF and acetonitrile. Reverse phase HPLC analysis ofthe reaction mixture reveals a very broad peak following the peak ofpeptide (24). Mass spectrometric analysis of the solid failed to giveany useful information. We assume that the solid material may resultfrom aggregation of the peptide, which has a concentration of 1 mM inthe reaction buffer. The clustered maleimide may serve as a cross-linkreagent for the non-covalently aggregating peptides, promoting furtherand eventually irreversible aggregations of the peptides. It waspreviously revealed that, in aqueous buffer (pH 7.0), T20 is monomericat the concentration below 10 μM, but exhibits complicatedmonomer/tetramer equilibrium and other aggregation patterns when theconcentration is above 20 μM. (11) We also observed that, under nativegel filtration conditions (PBS, pH 7.2) and at 0.5 mM, both T20 and thesynthetic peptide (24) appear as a series of broad peaks that havemolecular weights corresponding to the oligomeric and polymeric forms ofthe parent peptides (data not shown), indicating that indeed thepeptides form aggregates at relatively high concentrations.

We eventually found that the ligation of peptide (24) and the maleimidecluster (7) can proceed very smoothly in a 1:1 acetonitrile-phosphatebuffer (pH 7.0) to give the desired tetravalent peptide (25)(FIG. 7). Inthe presence of high concentration of acetonitrile, the peptide couldexist in monomeric form. The ligation is rapid and highly efficient.HPLC monitoring indicates that the reaction is actually finished within30 min. The tetravalent peptide (25) was purified in 82% yield byreverse phase HPLC, and the purity and identity of the product wasconfirmed by analytic HPLC and ES-MS, respectively.

Similarly, ligation of the peptide (24) to a different template, thetetravalent maleimide cluster (10), under the same reaction conditionsas described for the synthesis of 25 gave the tetravalent gp41 peptide(26) in 84% yield after preparative HPLC (FIG. 7).

EXAMPLE 8

Synthesis of Cholic Acid-Based Maleimide Clusters

Cholic acid is a rigid, amphiphilic steroid. The hydrophilic facecontains 3 hydroxyl groups at defined spatial orientations at 3α, 7α,and 12α positions. Therefore, cholic acid and other bile acids have beenused as scaffolds for combinatorial chemistry (Madder et al, 2002; Zhou,et al, 2000) and for the development of antimicrobial agents (Guan, etal, 2000). We have exploited the potential of chloic acid formultivalent peptide construction through the synthesis of cholicacide-based maleimide clusters, the general structures of which wereshown in FIG. 8.

Through selective chemical transformations of functional groups onchloric acid, several novel maleimide clusters were synthesized, inwhich the maleimide functionality was installed in a defined spatialorientation. This is particularly important because the orientation ofthe maleimide groups will determine the spatial orientation of thepeptide chains attached. The synthesis was summarized in FIG. 9.Briefly, three amine functionalities were selectively introduced at thepositions of the 3 hydroxyl groups in cholic acid 27 to give compound32. Then three maleimide functionalities were attached to afford themaleimide clusters 33 and 34, with long and short spacers, respectively.Similarly, a bromoacetyl group was introduced at each amino group incompound 32 to give the bromoacetyl derivative 35 (FIG. 9), which wasprepared for the comparison of the efficiency in ligation.

EXAMPLE 9

Ligation of Peptides with the Cholic Acid-Based Maleimide Clusters andthe Comparison with the Corresponding Bromoacetyl Derivative

Similar to the peptide ligation with the carbohydrate-based clusters,the peptide ligation of the cholic acid-based clusters was found to behighly efficient. As shown in FIG. 10, three different peptides werechosen and tested for the ligation. These include the HIV inhibitorDP178 (P37C), a T-helper epitope from tetanus toxoid (830-844), and aminimum epitope sequence ELDKWA (SEQ ID NO: 4) for HIV-neutralizingantibody 2F5. In the case of the T-helper sequence, a tetra-peptidespacer GSSS was introduced at the N-terminus to increase the aqueoussolubility of the otherwise hydrophobic T-helper epitope. Regardless thelength and complexity of the peptides, the peptide ligation to thecholic acid-based maleimide clusters gave the desired multivalentpeptide clusters (36a, 36b, 37a, 37b, and 37c) in very high yields (FIG.10).

However, when the bromoacetyl template 35 was used, no ligation productcould be obtained for the long peptide P37C. In the case of simple,short peptide ELDKWAC (SEQ ID NO: 1), the yield of the desired ligationproduct 38 was isolated in only 35% yield under optimal ligationconditions, together with mono- and di-substituted by-products. Theresults clearly show that the maleimide clusters are superior to otherfunctionalized templates for multivalent peptide assembly.

EXAMPLE 10

Conformational Studies of the Synthetic Multivalent Peptides Using CDSpectroscopy.

One application of the scaffolds is to induce special conformations ofpeptides. The circular dichroism (CD) spectra of the syntheticmultivalent gp41 peptide (MVP-1) and peptide DP178 were recorded andcompared (FIG. 11). It was shown that the multivalent assembling has asignificant impact on the peptide conformation, leading to a greatincrease of the α-helical content for the peptide. Based on [θ]₂₂₂ andthe proposed formula (Lyu et al, 1991), the content of α-helix was 18%for peptide DP178, which is consistent with the value reported inliterature (Lawless et al, 1996), whereas the α-helix content for MVP-1is 40%. The results suggest that the novel scaffold approach not onlyallows novel spatial presentation of the gp41 peptides, but also impactsthe conformations of the peptides.

Similarly, we measured the circular dichroism (CD) spectra of the cholicacid-based multivalent peptides (36a and 37b). The or-helix contents of36a and 37b were found to be 41 and 36%, respectively. In other words,the template-assembled peptides form a three-α-helix structure. Again,the results indicate that the spatially defined maleimide clusters areuseful templates for constructing suitable ax-helix bundles, whichshould be valuable for de novo artificial protein design.

EXAMPLE 11

Immunization Studies

We have synthesized novel template-assembled multivalent peptides tomimic the transition state structures of gp41, using peptide DP178(i.e., T20) as the model peptide. These multivalent peptides areotherwise difficult to obtain by other ligation methods. A preliminaryimmunization with MVP-1, which contains 4 strands of peptide DP178, wasconducted in mice. Same quantity (10 μg each) of the synthetic MVP-1 andthe single peptide DP178 itself were used to immunize/boost mice inorder to evaluate and compare their immunogenicity. The antibodyresponses were evaluated with ELISAs and the results were shown in FIG.4. The data clearly indicate that the multivalent gp41 peptide MVP-1 ishighly immunogenic. It was shown that the multivalent gp41 peptide MVP-1was able to elicit unusually high titers (over 1 to one million) ofantibody responses in the absence of any adjuvant (FIGS. 12 & 13). Sincenovel conformational epitopes may be induced by the rigid template-basedassembling (as revealed by the CD spectroscopic studies), the novelmultivalent peptides may elicit antibodies targeting nativeconformations found in HIV-1 and other pathogenic proteins, offering agreat potential for novel vaccine development. Moreover, when a T-helperepitope is incorporated in the construct, no additional adjuvants arerequired for raising high titers of specific antibodies. Anadjuvant-free, fully synthetic vaccine is a huge benefit in vaccinedevelopment and is of particular significance in terms of clinic trialsin the future.

Experimental Procedures

Fmoc-protected amino acids were purchased from Novabiochem. HATU, DIPEAand Fmoc-PAL-PEG-PS were purchased from Applied Biosystems. HPLC gradeacetonitrile was purchased from Fisher Scientific. DMF was purchasedfrom B & J Biosynthesis. All other chemicals were purchased fromAldrich/Sigma and used as received. ¹H NMR spectra were recorded on QE300 with Me₄Si (δ 0) as the internal standard. The ES-MS spectra weremeasured on a Waters ZMD mass spectrometer. Analytical TLC was performedon glass plates coated with silica gel 60 F₂₅₄ (E. Merck). Carbohydrateswere detected by charring with 10% ethanolic sulfuric acid. Amines weredetected by ninhydrin spraying. Flash chromatography was performed onsilica gel 60 (200-400 mesh, EM Science). Gel filtration was carried outon SEPHADEX G-15 (Pharmacia) using de-ionized water as the eluent.Photo-addition reactions were carried out in a quartz flask under N₂.Analytical HPLC was carried out with a Waters 626 HPLC instrument on aWaters NOVA-PAK C18 column (3.9×150 mm) at 40° C. The column was elutedwith a linear gradient of acetonitrile (10-90%) containing 0.1% TFA in25 min at a flow rate of 1 ml/min. Peptides were detected by UVabsorbance at 214 and/or 280 nm. Preparative HPLC was performed with aWaters 600 HPLC instrument on a Waters C18 column (SYMMETRY300, 19×300mm) and/or on a DELTA-PAK C18 column [DELTA-PAK RCM 2×(2.5×10 cm)]. Thecolumn was eluted with a linear gradient of acetonitrile (10-60%)containing 0.1% TFA at a flow rate of 10 ml/min. The peptides weredetected at 214 and/or 280 nm. Purified peptides were lyophilized andkept under nitrogen in a freezer (−20° C.).

The UV absorbance was run on Beckman DU 640 spectrophotometer. CD'sspectrums were measured on Jasco-810 (CD-ORD) spectropolarimeter usingquartz cuvette of 1 mm path length. The measurement temperature was 25°C.

Ethyl 2,3,4,6-tetra-O-allyl-α-D-galactopyranoside (2). A solution ofethyl α-D-galactopyranoside 1 (416.4 mg, 2.0 mmol) in anhydrous DMF (5mL) was added dropwise to a stirred suspension of sodium hydride (60%dispersion in mineral oil, 960.0 mg, 24.0 mmol) in anhydrous DMF (15mL). After 30 min, the reaction mixture was cooled to 0° C., and allylbromide (2.90 g, 24.0 mmol) was added dropwise. The resulting mixturewas stirred for 1 h at 0° C., and 3 h at room temperature. After themixture was cooled in an ice-bath, excess sodium hydride was quenched byslow addition of methanol (5 mL). The volatiles were evaporated todryness, and then the residue was mixed with ethyl acetate (150 mL) andwashed with brine (3×30 mL). The organic phase was dried over Na₂SO₄,filtered, and concentrated. The oily residue was purified by flashchromatography (hexane/EtOAc 85:15) to afford compound 2 (690.0 mg, 94%)as a colorless oil; R_(f) 0.65 (hexane/EtOAc 7:3); ¹H-NMR (300 MHz,CDCl₃/TMS): δ6.05-5.84 (m, 4H, OCH₂CH═CH₂), 5.36-5.10 (m, 8H,OCH₂CH═CH₂), 4.94 (d, 1H, J=3.7 Hz, H-1), 4.39 (dd, 1H, J=12.6, 5.6 Hz,OCHHCH═CH₂), 4.30-3.98 (m, 7H, OCH₂CH═CH₂), 3.93 (dd, 1H, J=7.1, 6.4 Hz,H-5), 3.84 (s, 1H, br, H-4), 3.83 (dd, 1H, J=9.8, 3.7 Hz, H-2),3.80-3.60 (m, 2H, H-3, and OCHHCH₃), 3.64-3.55 (m, 2H, H-6 and OCHHCH₃),3.52 (dd, 1H, J=9.0, 6.1 Hz, H-6′), 1.24 (t, 3H, J=7.1 Hz, OCH₂CH₃);ES-MS: 391.36 (M+Na)⁺, 323.32 (M-OEt)⁺, 207.14 (M+2Na)²⁺.

Ethyl 2,3,4,6-tetra-O-(3-hydroxypropyl)-α-D-galactopyranoside (3). To astirred solution of 2 (230.0 mg, 0.62 mmol) in dry THF (10 mL) was addeddropwise a solution of 9-BBN in THF (0.5 M, 10.0 mL, 5.0 mmol) at roomtemperature. The reaction mixture was heated under reflux for 2 h, andexcess of 9-BBN was then destroyed by dropwise addition of water at 0°C. The hydroboration mixture was oxidized by treatment with 3 M aqueousNaOH (11.0 mL) and 30% H₂O₂ solution (11.0 mL) at 0° C., followed bystirring overnight at room temperature. The mixture was saturated withK₂CO₃, and the phases were separated. The aqueous phase was extractedwith THF 2×30 mL) and the combined organic phases were dried overNa₂SO₄, filtered, and concentrated. The oily residue was purified bycolumn chromatography (ethyl acetate/Methanol 8:2) to yield tetraol 3(231.0 mg, 84%) as a colorless oil; R_(f) 0.49 (EtOAc/MeOH 7:3); ¹H-NMR(D₂O): δ5.10 (d, 1H, J=3.4 Hz, H-1), 4.02 (dd, 1H, J=5.8, 4.8 Hz,OCHHCH₂CH₂OH), 3.94 (s, 1H, br, H-4), 3.86-3.50 (m, 22H, H-2, H-3, H-5,H-6, H-6′, OCH₂CH₂CH₂OH, OCH₂CH₂CH₂OH and OCH₂CH₃), 1.86-1.75 (m, 8H,OCH₂CH₂CH₂OH), 1.19 (t, 3H, J=7.1 Hz, OCH₂CH₃); ES-MS: 463.45 (M+Na)⁺,441.47 (M+H)⁺, 395.43 (M-OEt)⁺, 243.26 (M+2Na)²⁺.

Ethyl 2,3,4,6-tetra-O-(3-azidopropyl)-α-D-galactopyranoside (5). To astirred solution of Ph₃P (1.00 g, 3.81 mmol) in dry DMF (3 mL) was addediodine (0.97 g, 3.81 mmol). After 10 min, a solution of tetraol 3 (210.0mg, 0.48 mmol) in DMF (2 mL) was added dropwise. The resulting mixturewas stirred for 2 h at room temperature and another 2 h at 80° C.Heating was then discontinued and the mixture was concentrated underreduced pressure to remove DMF. The residue was purified by flashchromatography (hexane/EtOAc 8:2) to give tetraiodide 4 (293.0 mg, 70%).The iodide 4 thus obtained was used immediately for the next step.Iodide 4: R_(f) 0.65 (hexane/EtOAc 7:3); ¹H-NMR (CDCl₃/TMS): 84.97 (d,1H, J=3.4 Hz, H-1), 3.96-3.83 (m, 2H, H-4, and OCHHCH₂CH₂I), 3.80˜3.47(m, 14H, H-2, H-3, H-5, H-6, H-6′, OCH₂CH₂CH₂I, and OCH₂CH₃), 3.36-3.24(m, 8H, OCH₂CH₂CH₂I), 2.20-1.98 (m, 8H, OCH₂CH₂CH₂I), 1.23 (t, 3H, J=7.1Hz, OCH₂CH₃).

A mixture of iodide 4 (260.0 mg, 0.30 mmol) and NaN₃ (1.24 g, 19.07mmol) in dry DMF (10 mL) was stirred overnight at room temperature. Themixture was evaporated at reduced pressure to dryness, and the residuewas partitioned in CH₂Cl₂ (100 mL) and water. The organic layer waswashed with brine (3×20 mL), dried (Na₂SO₄), filtered, and concentrated.The oily residue was purified by flash chromatography (hexane/EtOAc 8:2)to provide tetraazide 5 (124.0 mg, 78%); R_(f) 0.41 (hexane/EtOAc 7:3);¹H-NMR (CDCl₃/TMS): 84.95 (d, 1H, J=3.4 Hz, H-1), 3.96-3.85 (m, 2H, H-4,and OCHHCH₂CH₂N₃), 3.80˜3.47 (m, 14H, H-2, H-3, H-5, H-6, H-6′,OCH₂CH₂CH₂N₃, and OCH₂CH₃), 3.47-3.35 (m, 8H, OCH₂CH₂CH₂N₃), 1.95-1.76(m, 8H, OCH₂CH₂CH₂N₃), 1.25 (t, 3H, J=7.1 Hz, OCH₂CH₃) ); ES-MS: 563.39(M+Na)⁺, 467.42 (M-OEt-N₂)⁺.

Ethyl 2,3,4,6-tetra-O-(3-aminopropyl)-α-D-galactopyranoside (6). Azide 5(61.0 mg, 0.11 mmol) was hydrogenated with Pd/C (10%, 10 mg) in methanol(5 mL) overnight at room temperature. The mixture was filtered through abed of CELITE, and the filtrate was then concentrated to give tetraamine6 (51.5 mg, 100%); ¹H-NMR (D₂O): δ5.10 (d, 1H, J=3.4 Hz, H-1), 4.06-3.98(m, 1H, OCHHCH₂CH₂NH₂), 3.96-3.92 (m, 1H, H-4), 3.90˜3.48 (m, 14H, H-2,H-3, H-5, H-6, H-6′, OCH₂CH₂CH₂NH₂, and OCH₂CH₃), 2.89-2.70 (m, 8H,OCH₂CH₂CH₂NH₂), 1.90-1.65 (m, 8H, OCH₂CH₂CH₂NH₂), 1.18 (t, 3H, J=7.1 Hz,OCH₂CH₃); ES-MS: 437.48 (M+H)⁺, 219.16 (M+2H)²⁺.

Ethyl 2,3,4,6-tetra-O-(3-maleimidopropyl)-α-D-galactopyranoside (7). Asolution of amine 6 (17.8 mg, 0.04 mmol) in 1 M aqueous solution ofNaHCO₃ (1 mL) was treated with methoxycarbonylmaleimide (37.94 mg, 0.24mmol) at 0° C. After 5 min, the mixture was diluted with water (1 mL)and acetonitrile (2 mL), and then stirred at room temperature for 4 h.After adding CH₂Cl₂ (50 mL), the organic layer was separated and washedwith brine (3×10 mL). The organic layer was then dried (Na₂SO₄),filtered, and concentrated. The oily residue was purified by flashchromatography (CH₂Cl₂/MeOH 95:5) to give maleimide 7 (23.2 mg, 76%);R_(f) 0.42 (CH₂Cl₂/MeOH 95:5); ¹H-NMR (CDCl₃/TMS): δ6.70 (s, 2H, CH═CH),6.69 (s, 4H, CH═CH), 6.68 (s, 2H, CH═CH), 4.98 (d, 1H, J=3.4 Hz, H-1),3.95-3.82 (m, 2H, H-4, and OCHHCH₂CH₂N), 3.80˜3.35 (m, 22H, H-2, H-3,H-5, H-6, H-6′, OCH₂CH₂CH₂N, OCH₂CH₂CH₂N, and OCH₂CH₃), 1.96-1.78 (m,8H, OCH₂CH₂CH₂N), 1.25 (t, 3H, J=7.1 Hz, OCH₂CH₃); ES-MS: 779.28(M+Na)⁺, 401.29 (M+2Na)²⁺.

Ethyl 2,3,4,6-tetra-O-(6-amino-3-thia-hexyl)-α-D-galactopyranosidetetrahydrochloride (8). To a solution of the tetra-O-allyl derivative 2(404.6 mg, 1.10 mmol) and AIBN (30.0 mg) in methanol (15 mL) in a Quartzflask was added cysteamine hydrochloride (1.50 g, 13.20 mmol). Afterbeing degassed by bubbling N₂ into solution for 30 min, the resultingmixture was stirred and irradiated (UV, 254 nm) under N₂. The reactionwas monitored by measuring the ¹H-NMR of a small portion of the reactionmixture, which was dried and deuterium-exchanged with D₂O beforerecording the NMR. During the progress of the reaction, the signals atδ5.10-6.05 (for the allyl groups) decreased and the new signals atδ2.68-2.90 (for SCH₂) increased. After 24 h, NMR indicated thedisappearance of the allyl signals. MeOH was then evaporated and theresidue was purified by gel filtration on a SEPHADEX G-15 column usingwater as the eluent. Fractions containing the product were pooled andlyophilized to give amine 8 (723.4 mg, 80%) as a colorless glass-likesolid; ¹H-NMR (D₂O): δ5.08 (d, 1H, J=3.4 Hz, H-1), 4.0-3.98 (m, 1H,OCHHCH₂CH₂S), 3.96-3.92 (m, 1H, H-4), 3.90˜3.48 (m, 14H, H-2, H-3, H-5,H-6, H-6′, OCH₂CH₂CH₂S, and OCH₂CH₃), 3.26˜3.14 (m, 8H, SCH₂CH₂NH₂HCl),2.90-2.82 (m, 8H, SCH₂CH₂NH₂HCl), 2.72-2.68 (m, 8H, OCH₂CH₂CH₂S),1.96-1.78 (m, 8H, OCH₂CH₂CH₂S) 1.20 (t, 3H, J=7.1 Hz, OCH₂CH₃); ES-MS:677.1 (M+H-4HCl)⁺, 339.1 (M+2H-4HCl)²⁺.

Ethyl 2,3,4,6-tetra-O-(6-maleimido-3-thia-hexyl)-α-D-galactopyranoside(9). A solution of amine 8 (23.9 mg, 29.0 μmol) dissolved in 1 M aqueoussolution of NaHCO₃ (1 mL) was treated with methoxycarbonylmaleimide(27.03 mg, 17.4 μmol) at 0° C. After 5 min, the mixture was diluted withwater (1 mL) and acetonitrile (2 mL), and then stirred at roomtemperature for 1 h. The mixture was diluted with CH₂Cl₂ (50 mL) andwashed with brine (3×10 mL). The organic phase was dried over Na₂SO₄,filtered and concentrated. The oily residue was purified by flashchromatography (CH₂Cl₂/MeOH 96:4) to give maleimide 9 (20.3 mg, 71%);R_(f) 0.45 (CH₂Cl₂/MeOH 96:4); ¹H-NMR (CDCl₃/TMS): δ6.72 (s, 8H, CH═CH),4.94 (d, 1H, J=3.4 Hz, H-1), 3.95-3.82 (m, 2H, H-4, and OCHHCH₂CH₂N),3.80˜3.45 (m, 22H, H-2, H-3, H-5, H-6, H-6′, OCH₂CH₂CH₂S, SCH₂CH₂N, andOCH₂CH₃), 2.90-2.60 (m, 16H, OCH₂CH₂CH₂SCH₂CH₂N), 1.96-1.82 (m, 8H,OCH₂CH₂CH₂S), 1.23 (t, 3H, J=7.1 Hz, OCH₂CH₃); ES-MS: 1019.49 (M+Na)⁺,951(M-OEt)⁺.

Ethyl2,3,4,6-tetra-O-[(6-(6-maleimidohexanamido)-3-thia-hexyl)]-α-D-galactopyranoside(10). A solution of amine 8 (138.6 mg, 0.17 mmol) in 1 M aqueoussolution of NaHFCO₃ (1.5 mL) was added dropwise to a stirred solution of6-maleimidohexanoic acid N-hydroxylsuccinimide ester (272.5 mg, 0.88mmol) in THF (3 mL), which was cooled with a bath of ice-water. Theresulting mixture was stirred for 1 h at 0-5° C. The mixture was thendiluted with CHCl₃ (70 mL) and washed with brine (3×10 mL). The organicphase was dried over Na₂SO₄, filtered, and concentrated. The oilyresidue was purified by flash chromatography (CH₂Cl₂/MeOH 95:5) to givemaleimide 10 (103.6 mg, 43%); R_(f) 0.62 (CH₂Cl₂/MeOH 9:1); ¹H-NMR(CDCl₃/TMS): δ 6.68 (s, 8H, CH═CH), 6.26-6.20 (m, 2H, NH), 6.12 (t, 1H,J=6.8 Hz, NH), 6.04 (t, 1H, J=6.8 Hz, NH), 4.92 (d, 1H, J=3.2 Hz, H-1),3.95-3.86 (m, 2H, H-4, and OCHHCH₂CH₂N), 3.80˜3.40 (m, 30H, H-2, H-3,H-5, H-6, H-6′, OCH₂CH₂CH₂S, SCH₂CH₂N, NCH₂(CH₂)₄ and OCH₂CH₃),2.75-2.60 (m, 16H, OCH₂CH₂CH₂SCH₂CH₂N), 2.18 (t, 8H, J=7.3 Hz,N(CH₂)₄CH₂), 1.96-1.82 (m, 8H, OCH₂CH₂CH₂S), 1.74-1.54 (m, 16H,NCH₂CH₂CH₂CH₂CH₂), 1.37-1.24 (m, 8H, N(CH₂)₂CH₂(CH₂)₂), 1.22 (t, 3H,J=7.1 Hz, OCH₂CH₃); ES-MS: 1450.01 (M+H)⁺, 725.56 (M+2H)²⁺.

(6-Amino-3-thia-hexyl)2,3,4,6-tetra-O-(6-amino-3-thia-hexyl)-β-glucopyranosidepentahydrochloride (12). To a solution of penta-O-allyl derivative 11(12)(628.5 mg, 1.65 mmol) and AIBN (50.0 mg) in methanol (15 mL) in aquartz flask was added cysteamine hydrochloride (2.80 g, 24.67 mmol).After being degassed by bubbling N₂ into the solution for 30 min, theresulting mixture was stirred and irradiated (UV, 254 nm) under N₂. Thereaction was monitored by ¹H-NMR, After 24 h when NMR showed thedisappearance of the allyl signals, the solvent removed by evaporationand the residue was subjected to gel filtration on a column of SEPHADEXG-15 using water as the eluent. Fractions containing the product werepooled and lyophilized to give amine 12 (1.25 g, 80%) as a colorlessglass-like solid; ¹H-NMR (D₂O): δ4.43 (d, 1H, J=7.8 Hz, H-1), 4.04-3.40(m, 13H, H-5, H-6, H-6′, OCH₂CH₂CH₂S), 3.40 (dd, 1H, J=8.5, 9.1 Hz,H-4), 3.36 (dd, 1H, J=9.1, 9.3 Hz, H-3), 3.26-3.16 (m, 10H,SCH₂CH₂NH₂HCl), 3.11 (dd, 1H, J=7.8, 9.3 Hz, H-2), 2.90-2.82 (m, 10H,SCH₂CH₂NH₂HCl), 2.72-2.64 (m, 10H, OCH₂CH₂CH₂S), 1.96-1.84 (m, 10H,OCH₂CH₂CH₂S); ES-MS: 766.1 (M+H-5HCl)⁺, 383.7 (M+2H-5HCl)²⁺.

[6-(6-maleimidohexanamido)-3-thia-hexyl]2,3,4,6-tetra-O-[6-(6-maleimidohexanamido)-3-thia-hexyl)-β-D-glucopyranoside(13). A solution of amine 12 (109.8 mg, 0.12 mmol) in 1 M aqueoussolution of NaHCO₃ (1.2 mL) was added dropwise to a stirred solution of6-maleimidohexanoic acid N-hydroxylsuccinimide ester (214.2 mg, 0.70mmol) in THF (3 mL) that was cooled with a bath of ice-water. Theresulting mixture was stirred for 1 h at 0-5° C., and then diluted withCHCl₃ (70 mL). The organic layer was separated and washed with brine(3×10 mL), dried (Na₂SO₄), filtered, and concentrated. The oily residuewas subjected to column chromatography (CH₂Cl₂/MeOH 95:5) to givepentamaleimide 13 (76.7 mg, 39%); R_(f) 0.35 (CH₂Cl₂/MeOH 95:5); ¹H-NMR(CDCl₃/TMS): δ6.69 (s, 10H, CH═CH), 6.28-6.15 (m, 5H, NH), 4.18 (d, 1H,J=7.8 Hz, H-1), 3.95-3.50 (m, 13H, H-5, H-6, H-6′, OCH₂CH₂CH₂S), 3.50(t, 10H, J=7.2 Hz, NCH₂(CH₂)₄), 3.50-3.38 (m, 10H, SCH₂CH₂N), 3.24-3.16(m, 2H, H-3, H-4), 3.04-2.96 (m, 1H, H-2), 2.70-2.58 (m, 20H,OCH₂CH₂CH₂SCH₂CH₂N), 2.18 (t, 10H, J=7.3 Hz, N(CH₂)₄CH₂), 1.92-1.78 (m,10H, OCH₂CH₂CH₂S), 1.74-1.54 (m, 20H, NCH₂CH₂CH₂CH₂CH₂), 1.38-1.24 (m,10H, N(CH₂)₂CH₂(CH₂)₂); ES-MS: 1733.16 (M+H)⁺, 866.84 (M+2H)²⁺.

Tetra-O-allyl pentaerythritol (15). A solution of pentaerythritol 14(0.68 g, 5.0 mmol) in anhydrous DMF (10 mL) was added dropwise to astirred suspension of sodium hydride (60% dispersion in mineral oil,2.01 g, 50.0 mmol) in anhydrous DMF (15 mL). After 30 min, the reactionmixture was cooled to 0° C., and then allyl bromide (4.84 g, 40.0 mmol)was added dropwise with a syringe. The resulting mixture was stirred for1 h at 0° C., and overnight at room temperature. After the mixture wascooled with an ice bath, the excess sodium hydride was quenched by theslow addition of methanol (5 mL). The volatile was evaporated todryness, and then the residue was mixed with ethyl acetate (150 mL) andwashed with brine (3×30 mL). The organic phase was dried over Na₂SO₄,filtered, and concentrated. The oily residue was purified through flashchromatography (hexane/EtOAc 97:3) to afford compound 15 (1.34 g, 91%)as a colorless oil; ¹H-NMR (300 MHz, CDCl₃/TMS): δ5.95-5.81 (m, 4H,OCH₂CH═CH₂), 5.30-5.11 (m, 8H, OCH₂CH═CH₂), 3.96 (m=s, 4H, OCH₂CH═CH₂),3.94 (m=s, 4H, OCH₂CH═CH₂), 3.46 (s, 8H, CH₂OCH₂CH═CH₂); ESI-MS: 319.32(M+Na)⁺, 297.31 (M+H)⁺.

Tetra-O-6-amino-3-thiahexyl pentaerythritol tetrahydrochloride (16). Toa solution of compound 15 (151.4 mg, 0.51 mmol) and AIBN (20.0 mg) inmethanol (10 mL) in a Quartz flask was added cysteamine hydrochloride(348.2 mg, 3.06 mmol). After being degassed by bubbling N₂ into solutionfor 30 min, the resulting mixture was stirred and irradiated (UV, 254nm) under N₂ for 24. The volatile was evaporated under reduced pressure,and the residue was then purified by gel filtration on SEPHADEX G-15using water as eluent. Fractions containing the product were pooled andlyophilized to give compound 16 (319.4 mg, 84%); ¹H-NMR (D₂O): δ3.56 (t,8H, J=6.1 Hz, CCH₂OCH₂CH₂CH₂SCH₂CH₂NH₂HCl), 3.40 (s, 8H,CCH₂OCH₂CH₂CH₂SCH₂CH₂NH₂HCl), 3.20 (t, 8H, J=6.6 Hz,CCH₂OCH₂CH₂CH₂SCH₂CH₂NH₂HCl), 2.83 (t, 8H, J=6.7 Hz,CCH₂OCH₂CH₂CH₂SCH₂CH₂NH₂HCl), 2.63 (t, 8H, J=7.2 Hz,CCH₂OCH₂CH₂CH₂SCH₂CH₂NH₂HCl), 1.85 (p, 8H, J=6.7 Hz,CCH₂OCH₂CH₂CH₂SCH₂CH₂NH₂HCl); ESI-MS: 627.26 (M+Na-4 HCl)⁺, 605.30(M+H-4 HCl)⁺, 303.18 (M+2H-4HCl)²⁺.

Tetra-O-6-(6-maleimidohexanamido)-3-thiahexyl pentaerythritol (17). Asolution of compound 16 (55.4 mg, 73.8 μmol) in 1 M aqueous solution ofNaHCO₃ (2 mL) was added dropwise to a stirred solution of N-succimidyl6-maleimidohexanoic acid ester (135.6 mg, 0.44 mmol) in THF (3 mL)cooled with a bath of ice-water. The resulting mixture was stirred for 1h at 0° C., and then diluted with ethyl acetate (60 mL) and washed withbrine (3×10 mL). The organic phase was dried over Na₂SO₄, filtered andconcentrated. The oily residue was purified through flash chromatographyto give compound 17 (47.0 mg, 47%); ¹H-NMR (CDCl₃/TMS): δ 6.89 (s, 8H,CH═CH), 6.12 (t, 4H, J=5.4 Hz, NH), 3.51 (t, 8H, J=7.2 Hz,CCH₂OCH₂CH₂CH₂S), 3.48-3.40 (m, 16H, SCH₂CH₂NH, NCH₂(CH₂)₄), 3.34 (s,8H, CCH₂OCH₂CH₂CH₂S), 2.65 (t, 8H, J=6.4 Hz, SCH₂CH₂NH), 2.58 (t, 8H,J=7.4 Hz, CCH₂OCH₂CH₂CH₂S), 2.18 (t, 8H, J=7.4 Hz,NHCOCH₂CH₂CH₂CH₂CH₂N), 1.81 (p, 8H J=6.8 Hz, OCH₂CH₂CH₂S), 1.72-1.54 (m,16H, NCH₂CH₂CH₂CH₂CH₂), 1.38-1.24 (m, 8H, N(CH₂)₂CH₂(CH₂)₂); ESI-MS:1399.98 (M+Na)⁺, 1377.97 (M+H)⁺, 689.75 (M+2H)²⁺.

Typical procedures for the allylation of cyclodextrins: Each of thecyclodextrins (2 mmol) was dissolved in DMF (15 mL) by heating andstirring. The clear solution was then added dropwise to a cooled (0° C.)suspension of sodium hydride (60% dispersion in mineral oil, 3 equiv.Per OH group in cyclodextrin) that was washed with dry hexane (2×10 ml)before being suspended in DMF (50 mL). After the suspension was stirredat 0-5° C. for 1 h, a solution of allyl bromide (3 equiv. Per OHfunction) in DMF (10 mL) was added dropwise. The resulting mixture wasfirst stirred at 0-5° C. for 1 h, then at 25° C. for 2-4 h. At thispoint, TLC showed the formation of a single product. The reaction wasquenched by addition of MeOH (5 mL). DMF and excess allyl bromide wereremoved by evaporation under a reduced pressure. The residue waspartitioned between ethyl acetate (200 mL) and water (30 mL). Theorganic layer was washed with brine (2×50 ml), dried over Na₂SO₄, andevaporated. Flash column chromatography of the residue on silica gelusing hexane-ethyl acetate (3:1, v/v) as the eluant gave the respectiveO-perallylated cyclodextrins.

Per-O-allyl-β-cyclodextrin (19): yield 91%; R_(f)=0.41 (25:1toluene-EtOH), and 0.31 (3:1 hexane-EtOAc); [α]₂₃ _(D)=+53° (c 0.5,CHCl₃), lit.₂₀ [α]₂₅ _(D)=+92° (c 0.5, CHCl₃); ¹H-NMR (CDCl₃/TMS):δ6.16˜6.05 (m, 7H, OCH₂CH═CH₂), 6.05˜5.92 (m, 14H, OCH₂CH═CH₂),5.44˜5.29 (m, 21H, OCH₂CH═CHH), 5.25 (d, 7H, J=3.4 Hz, H-1), 5.24˜5.14(m, 21H, OCH₂CH═CHH), 4.57 (dd, 7H, J=12.1, 5.3 Hz, OCHHCH═CH₂), 4.35(dd, 7H, J=12.1, 5.6 Hz, OCHHCH═CH₂), 4.30˜4.17 (m, 14H, OCHHCH═CH₂),4.17˜4.04 (m, 14H, OCHHCH═CH₂), 3.99 (dd, 7H, J=10.5, 2.4 Hz, H-6),3.92˜3.64 (m, 21H, H-3, H-4 and H-5), 3.67 (d, 7 H J=10.5 Hz, H-6′),3.43 (dd, 7H, J=9.4, 3.4 Hz, H-2); ¹³C NMR (CDCl₃): 138.84 (OCH₂CH═CH₂),138.00 (OCH₂CH═CH₂), 137.52 (OCH₂CH═CH₂), 119.36 (OCH₂CH═CH₂), 119.30(OCH₂CH═CH₂), 118.19 (OCH₂CH═CH₂), 101.33 (C-1), 82.64 (C-3), 81.86(C-2), 81.68 (C-4), 77.07 (OCH₂CH═CH₂), 74.79 (OCH₂CH═CH₂), 74.75(OCH₂CH═CH₂), 73.63 (C-5), 71.70 (C-6); ES-MS: 1994.4 (M-3H₂+Na)⁺,1977.4 (M+H)⁺, 1008.4 (M-3H₂+2Na)²⁺, 989.4 (M+2H)²⁺.

Per-O-(3-((hydrochloride amino)ethylthio)propyl)-β-cyclodextrin (20). Toa solution of 19 (213.0 mg, 0.11 mmmol) and AIBN (20.0 mg) in MeOH (10mL) in a quartz flask was added cysteamine hydrochloride (771.4 mg, 6.79mmol). After degassed by bubbling N₂ into the solution for 30 min, theresulting mixture was stirred and irradiated (UV 254 nm) under N₂atmosphere. The progress of the reaction was monitored with ¹H NMR.After 5 days, ¹H NMR showed the disappearance of allyl proton signals.The volatiles were evaporated under reduced pressure, and the residuewas purified by gel filtration on SEPHADEX G-15 using water as theeluent. Fractions containing the product was pooled and lyophilized togive the polyamine hydrochloride 20 (317.0 mg, 68%) as a colorlessglass-like solid: [α]₂₃ _(D)=+22° (c 0.5, H₂O); ¹H NMR (D₂O): δ5.35˜5.10(m, 7H, H-1), 4.20˜3.50 (m, 70H, OCH₂CH₂CH₂S—, H-3, H-5, H-6′ and H-6),3.48˜3.32 (m, 14H, H-2 and H-4), 3.30˜3.14 (m, 42H, —CH₂NH₂HCl),3.00˜2.84 (m, 42H, —SCH₂CH₂NH₂HCl), 2.84˜2.60 (m, 42H,—CH₂SCH₂CH₂NH₂HCl), 2.10˜1.85 (m, 42H, —CH₂CH₂CH₂S); ¹³C NMR (D₂O):101.00˜99.00 (C-1), 84.00˜80.00 (C-2, C-3, and C-4), 77˜73.00(C-5, andC-6), 73.00˜70.00 (˜OCH₂CH₂CH₂S), 41.22 (SCH₂CH₂NH₂HCl), 31.92(—OCH₂CH₂CH₂S), 31.23 (SCH₂CH₂NH₂HCl), 30.15 (OCH₂CH₂CH₂S).

Per-O-6-(6-maleimidohexanamido)-3-thiahexyl)-β-cyclodextrin (21). To thestirred solution of compound 20 (44.6 mg, 10.0 μmol) and N-succimidyl6-maleimidohexanoic acid ester (129.5 mg, 0.42 mmol) in methanol (2 mL)was added 1 M aqueous solution of NaHCO₃ (1.0 mL) at 0-5° C. Theresulting mixture was stirred for 1 h at 0-5° C., and then diluted withChloroform (60 mL) and washed with water (3×10 mL). The organic phasewas dried over Na₂SO₄, filtered and concentrated. The oily residue waspurified through flash chromatography to give compound 21 (38.6 mg,51%); ¹H-NMR (DMSO-d₆): δ7.96-7.86 (m, 21H, SCH₂CH₂NH), 6.97 (s, 42H,CH═CH), 5.11-5.00 (m, 7H, H-1), 4.00-3.05 (m, 168H, H-2, H-3, H-4, H-5,H-6, H-6′, OCH₂CH₂CH₂SCH₂CH₂NH, NH(O)CCH₂CH₂CH₂CH₂CH₂N), 2.65-2.38 (m,84H, OCH₂CH₂CH₂SCH₂CH₂NH), 2.08-1.97 (m, 42H, NH(O)CCH₂CH₂CH₂CH₂CH₂N),1.88-1.65 (m, 42H, OCH₂CH₂CH₂S), 1.60-1.35 (m, 84H, NCH₂CH₂CH₂CH₂CH₂),1.30-1.10 (m, 42H, N(CH₂)₂CH₂(CH₂)₂).

Peptide synthesis. All peptides used were synthesized on a PioneerPeptide Synthesizer (Applied Biosystems, Foster City, Calif.) usingFmoc-chemistry on Fmoc-PAL-PEG-PS resin. 4-Fold excess ofN_(α)-Fmoc-protected amino acids were used for each coupling andHATU/DIPEA were used as the coupling reagents. The N-terminus of thepeptide was capped with an acetyl group. Cleavage of the peptide fromthe resin with simultaneous deprotection was performed withTFA:thioanisole:EDT:anisole ((90:5:3:2)(cocktail R), and the crudepeptide was precipitated with cold ether. Purification of the peptidewas carried out by preparative HPLC as described in the general methods.Using the methods, the following peptides were synthesized and purified.

DP178 containing a cysteine at the C-terminus (P37C),Ac-YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFC-NH2 (SEQ ID NO: 3) retentiontime (tR), 15.8 min; ESI-MS, 1532.84 (M+3H) 3+, 1149.80 (M+4H) 4+,920.12 (M+5H)s+.

The minimum epitope of 2F5 (P7C), Ac-ELDKWAC-NH2 (SEQ ID NO: 1)retention time (tR), 13.5 min; ESI-MS, 905.53 (M+H) 1+, 453.42 (M+2H)2+.

The T-helper epitope derived from tetanus toxoid (830-844),CGSSSQYIKANSKFIGITEL-NH2 (SEQ ID NO: 5) retention time, 13.88 min;ESI-MS: 1431.68 (M+2H) 2+, 1073.90 (M+3H) 3+, 716.42 (M+4H) 4+.

Tetravalent peptide (22). To a solution ofAc-Glu-Leu-Asp-Lys-Trp-Ala-Cys-NH2 (SEQ ID NO: 1)(peptidel)(15 mg, 16.6mol) in phosphate buffer (50 mM, pH 7.0, 2.0 mL) and acetonitrile (2.0mL) was added dropwise a solution of maleimide 10 (3.8 mg, 2.6 llmol) inDMF (100 μL). After shaken at room temperature for 1 h, the reactionmixture was lyophilized. The residue was purified by RP-HPLC asdescribed in general methods, giving the tetravalent peptide 22 (12 mg,91%) as a white powder. ES-MS of 22: 1691.6 (M+3H) 3+, 1269.2 (M+4H) 4+,1015.8 (M+5) 5+.

Tetravalent peptide (23). To a solution ofAc-Cys-Glu-Leu-Asp-Lys-Trp-Ala-NH2 (SEQ ID NO: 2)(peptide2)(10 mg, 11mmol) in phosphate buffer (50 mM, pH 7.0, 1.5 mL) and acetonitrile (1.5mL) was added a solution of maleimide 10 (2.67 mg, 1.83 p, mol) in DMF(210 pal). After 1 h at room temperature, the reaction mixture waslyophilized and the residue was purified by preparative HPLC asdescribed in General Methods to afford the tetravalent peptide 23 (8.2mg, 88%). ES-MS of 23: 1692.0 (M+3H) 3+, 1270.1 (M+4H) 4+, 1016.1 (M+5)5+.

Peptide (24). The peptide 24 was synthesized on a PIONEER peptidesynthesizer (Applied Biosystems, Foster City, Calif.) usingFmoc-chemistry on Fmoc-PAL-PEG-PS resin. 4-Fold excess ofN^(α)-Fmoc-protected amino acids were used for each coupling andHATU/DIPEA were used as the coupling reagents. The N-terminus of thepeptide was capped with an acetyl group. Cleavage of the peptide fromthe resin with simultaneous deprotection was performed withTFA:thioanisole:EDT:anisole (90:5:3:2)(cocktail R), and the crudepeptide was precipitated with cold ether. Purification of the peptidewas carried out by preparative HPLC as described in the general methods.The purified 24 appeared as a single peak on analytic HPLC. Under theanalytic conditions (General Methods), the peptide was eluted at 15.84min. ES-MS of 24: 1532.84 (M+3H)³⁺, 1149.80 (M+4H)⁴⁺, 920.12 (M+5H)⁵⁺.

Tetravalent peptide (25). To a solution of peptide 24 (20.0 mg, 4.35μmol) in phosphate buffer (50 mM, pH 7.0, 3.0 mL) and acetonitrile (3.0mL) was added dropwise a solution of maleimide 7 (0.52 mg, 0.69 μmol) inDMF (104 μL). After shaken at room temperature for 2 h, the reactionmixture was lyophilized. The residue was purified by RP-HPLC asdescribed in general methods, giving the tetravalent peptide 25 (10.7mg, 82%) as a white powder. Analytical HPLC showed that the purified 25showed up at 16.47 min as a single peak. ES-MS of 25: 1914.75(M+10H)¹⁰⁺, 1741.00 (M+11H)¹¹⁺, 1596.01 (M+12H)¹²⁺, 1473.36 (M+13H)¹³⁺,1368.29 (M+14H)¹⁴⁺, 1276.76 (M+15H)¹⁵⁺.

Tetravalent peptide (26). To a solution of peptide 24 (20.0 mg, 4.35μmol) in phosphate buffer (50 mM, pH 7.0, 3.0 mL) and acetonitrile (3.0mL) was added a solution of maleimide 10 (1.05 mg, 0.72 μmol) in DMF(210 μL). After 2 h at room temperature, the reaction mixture waslyophilized and the residue was purified by preparative HPLC asdescribed in General Methods to afford the tetravalent peptide 26 (12mg, 84%). Analytical HPLC showed a single peak for 26 with a retentiontime of 16.85 min. ES-MS of 26: 1653.72 (M+12H)¹²⁺, 1526.60 (M+13H)¹³⁺,1417.63 (M+14H)¹⁴⁺, 1323.20 (M+15H)¹⁵⁺.

3α, 7α, 12α, 24-tetra-Hydroxcholane (28). To a suspension of LiAlH₄(3.85 g, 100 mmol) in 100 mL dry THF was added dropwise a solution ofmethyl ester 27b (15.03 g, 35 mmol) in 200 mL dry THF in 30 min undernitrogen with vigorously stirring in an ice bath. The react mixture wasthen stirred at room temperature overnight. Celite (3.00 g) was added tothe sluggish mixture. The precipitate was removed via filtration andwashed with hot methanol. The filtrate was concentrated under vacuum to80 mL. Another 150 mL acetone was added to recrystallize. Afterfiltration and washing with acetone, 28 was obtained as a whit solid(13.20 g, 98%). ¹H NMR (500 MHz, CDCl₃/TMS) δ 3.96 (bs, 1H, HO—C(12)),3.82 (bs, 1H, HO—C(7)), 3.79 (d, 2H, J=2.5 Hz, H₂—C(24)), 3.25 (b, 1H,HO—C(24)), 3.21 (m, 1H, H—C(12)), 3.15 (m, 1H, H—C(7)), 3.07 (m, 1H,H—C(3)), 2.15-1.10 (series of multiplet, 25H), 0.92 (d, 3H, J=6.5 Hz,H₃C—C(20)), 0.89 (s, 3H, H₃C—C(10)), 0.65 (s, 3H, H₃C—C(13)); ES-MS:789.47 (2×M+H)⁺, 395.58 (M+H)⁺.

3α, 7α, 12α-tri-hydroxyl-24-trityloxyl-5β-cholane (29). Compound 28(6.23 g, 15.8 mmol) was mixed with trityl chloride (6.60 g, 23.7 mmol)and dissolved in 100 mL DMF. To this mixture was added Et₃N (10 mL) andDBU (0.90 g, 5.9 mmol) with stirring at room temperature. After 24 hoursreaction, 400 mL water was added, and the mixture was stirred vigorouslyfor 2 hours. The precipitate was collected via filtration and washedwith acetone. The crude product was re crystallized in 50 mL acetone,after filtration and washing cold acetone, a white solid 29 was provided(9.35 g, 93%). ¹H NMR (300 MHz, CDCl₃/TMS) δ 7.43 (d, 6H, J=7.3 Hz,phenyl H-2, H-6), 7.29-7.18 (m, 9H, phenyl H-3, H-4, H-5), 3.96 (bs, 1H,H—C(12)), 3.82 (bs, 1H, H—C(7)), 3.41 (m, 1H, H—C(3)), 3.00 (m, 2H),2.92 (s, 2H), 2.87 (s, 2H), 2.19 (m, 2H), 2.04 (s, 1H), 1.98-1.00(series of multiplet, 16H), 0.96 (d, 3H, J=6.1 Hz, H₃C—C(20)), 0.86 (s,3H, H₃C—C(10)), 0.64 (s, 3H, H₃C—C(13)).

3α, 7α, 12α-tri-pentaoxyl-24-trtyloxyl-5β-cholane (30). Sodium hydride(60% in mineral oil, 2.00 g, 29.2 mmol) was added to a solution ofcompound 29 (3.12 g, 4.8 mmol) in mL dry THF in portion under N₂, andstirred for 2 hours at room temperature. The mixture was heated to 70°C., allyl iodide (4.98 g, 30.0 mmol) was added dropwise in 10 min andthen the mixture was stirred for 4 hours. After cooled to roomtemperature, 30 mL ethyl acetate was added to dilute, followed by adding20 mL water. The organic layer was washed with H₂O (2×20 mL) and brine,and dried over anhydrous Na₂SO₄. After removing the solvent under vacuumthe residue was purified on flash column chromatography with elution ofhexane/ethyl acetate (90:10). A pale yellow syrup-like 30 was obtained(3.49 g, 96%). ¹H NMR (300 MHz, CDCl₃/TMS) δ 7.44 (d, 6H, J=7.1 Hz,benzyl H-2, H-6), 7.29-7.20 (m, 9H, benzyl H-3, H-4, H-5), 5.88 (m, 3H,HC═CH₂), 5.23 (m, 3H, HHC═CHC), 5.11 (m, 3H, HHC═CHC), 4.13 (m, 4H,OH₂CHC═CH₂, (C-7, C-12)), 3.99 (bs, 2H, OH₂CHC═CH₂, C-3), 3.88-3.50(series of multiplet, 6 H), 3.35 (t, 2H, J=8.3 Hz, H₂—C(24)), 3.32 (bs,1H, H—C(12)), 3.16 (m, 1H, H—C(7)), 3.03 (m, 1H, H—C(3)), 3.07 (m, 1H,H—C(3)), 2.30-1.02 (series of multiplet, 18H), 0.92 (d, 3H, J=6.0 Hz,H₃C—C(20)), 0.89 (s, 3H, H₃C—C(10)), 0.66 (s, 3H, H₃C—C(13)).

3α, 7α, 12α-tri-pentaoxyl-24-hydroxyl-5β-cholane (31). p-Toluenesulfonicacid (45.0 mg, 0.23 mmol) was added to a solution of compound 30 (1.20g, 1.6 mmol) dissolved in mixture of 40 mL DCM and 10 mL methanol. Themixture was stirred at room temperature overnight, after diluted withDCM (20 mL) the mixture was washed with 0.5 NaHCO₃ (15 mL), H₂O (2×15mL) and brine. The organic layer was dried over anhydrous Na₂SO₄,concentrated under vacuum. The residue was separated by flash columnchromatography with elution of hexane/ethyl acetate (70:30) to give 31as a colorless syrup (0.83 g, 100%). ¹H NMR (500 MHz, CDCl₃/TMS) δ 5.91(m, 3H, HC═CH₂), 5.25 (m, 3H, HHC═CHC), 5.11 (m, 3H, HHC═CHC), 4.07 (d,2H, J=12.5 Hz, OH₂CHC═CH₂, (C-12)), 4.00 (d, 2H, J=5.5 Hz, OH₂CHC═CH₂,(C-7)), 3.77 (dt, 1H, J=7.5, 5.0 Hz, HH—C(24)), 3.71 (ddd, 1H, J=6.0,4.5, 1.5 Hz, HH—C(24)), 3.61 (b, 2H, OH₂CHC═CH₂, (C-3)), 3.54 (bs, 1H,H—C(12)), 3.32 (d, 1H, J=2.5 Hz, H—C(7)), 3.13 (m, 1H, H—C(3)), 2.26(dd, 1H, J=13.0, 12.0 Hz, H—C(11)), 2.18 (ddd, 1H, J=8.0, 8.0, 4.0 Hz,H—C(8)), 2.01 (dd, 1H, J=10.0, 9.5 Hz, H—C(11)), 1.87-0.96 (series ofmultiplet, 24H), 0.92 (d, 3H, J=6.5 Hz, H₃C—C(20)), 0.89 (s, 3H,H₃C—C(10)), 0.65 (s, 3H, H₃C—C(13)); ¹³C NMR (500 MHz, CDCl₃/TMS)136.26, 116.45, 115.69, 95.00, 80.98, 79.34, 75.07, 68.58, 69.47, 68.93,63.93, 63.90, 46.58, 46.55, 42.81, 42.25, 42.23, 40.03, 35.76, 35.62,35.60, 35.24, 35.23, 32.02, 29.69, 29.11, 28.22, 27.86, 27.69, 23.44,23.22, 18.03, 12.79; ESI-MS calcd. For C₃₃H₅₅O₄ ⁺ (M+H)⁺: 515.40; found:1029.80 (2×M+H)⁺, 515.57 (M+H)⁺, 457.52 (M−58+H)⁺, 399.47 (M−2×58+H)⁺,341.47 (M−3×58+H)⁺.

3α, 7α, 12α-tri-(6-Amino-3-thia-hexy-oxyl)-24-hydroxyl-5β-cholane (32).To a mixture of compound 31 (300 mg, 0.58 mmol) and 2-aminoethanethiolhydrochloride (397 mg, 3.50 mmol) in 20 mL methanol contained by aQuartz flask was added ABIN (9.4 mg, 0.057 mmol). The solution wasdegassed by bubbling N₂, and irradiated by UV (254 nm) with stirringunder N₂ overnight. 30 mL DCM was added to dilute, and the reactionmixture was washed with 0.5 M NaHCO₃ (2×10 mL), H₂O (2×5 mL) and brine.The organic layer was dried over anhydrous NaSO₄, concentrated undervacuum. The crude residue was separated over flash chromatography withgradient elution of ethyl acetate/methanol (0:100 to 100:0), to give 32as a glass-like solid (415 mg, 96%). ¹H NMR (500 MHz, CDCl₃/TMS) δ 3.74(m, 1H, H—C(24)), 3.68 (m, 1H, H—C(24)), 3.61 (m, 2H, OCH₂CCH₂CH₂S),3.56 (bs, 2H, OCH₂CCH₂CH₂S), 3.53 (t, 2H, J=7.0 Hz, OCH₂CCH₂CH₂S), 3.35(m, 7H, CH₂CH₂NH₂, HO—C(24)), 3.22 (m, 1H, H—C(12)), 3.16 (m, 7H,NCH₂CH₂S, H—C(7)), 3.06 (m, 1H, H—C(3)), 2.86 (m, 6H, OCH₂CH₂CH₂S), 2.74(m, 3H, OCH₂CHHCH₂S), 2.68 (t, 3H, J=7.0 Hz, OCH₂CHHCH₂S), 2.20-1.05(series of multiplet, 32H), 0.98 (d, 3H, J=6.0 Hz, H₃C—C(20)), 0.94 (s,3H, H₃C—C(10)), 0.72 (s, 3H, H₃C—C(13)); ¹³C NMR (500 MHz, CDCl₃/TMS)80.82, 79.95, 76.15, 66.19, 65.82, 62.47, 47.36, 46.46, 46.29, 42.86,42.10, 40.64, 39.87, 38.91, 38.86, 35.72, 35.45, 35.12, 34.82, 32.11,30.14, 29.93, 29.83, 28.93, 28.79, 28.61, 28.54, 28.47, 28.33, 28.00,27.65, 27.59, 23.32, 22.72, 22.16, 17.52, 17.13, 14.47, 11.75; ESI-MScalcd. for C₃₉H₇₆N₃O₄S₃ ⁺ (M+H)⁺: 746.49; found: 746.58 (M+H)⁺, 373.98(M+2H)²⁺.

3α, 7α,12α-tri-[6-(6-maleimidohexanamido-3-thia-hexy-oxyl)]-24-hydroxyl-5β-cholane(33). To a solution of free amine 32 (7.2 mg, 9.6 μmol) in 10 mL DCM wasadded 6-maleimidohexanoic acid N-hydroxylsuccinimide ester (30 mg, 96μmol). The mixture was stirred at room temperature for 3 hours, TLCshowed no more free amine remained in the reaction mixture. The solventwas removed under vacuum, and the residue was purified through flashchromatography with elution of ethyl acetate/methanol (95:5) to give 33as a colorless oil was provided (9.6 mg, 76%). ¹H NMR (500 MHz,CDCl₃/TMS) δ 6.72 (s, 6H, HC═CH), 6.61 (t, 1H, J=6.5 Hz, HN), 6.48 (t,1H, J=6.5 Hz, HN), 6.19 (t, 1H, J=6.5 Hz, HN), 3.59 (td, 2H, J=13.0, 6.5Hz, H₂—C(24)), 3.52 (t, 6H, J=7.0 Hz, OCH₂CCH₂CH₂S), 3.47 (bs, 6H,CH₂CH₂NCO) 3.43 (m, 6H, NCH₂CH₂S), 3.25 (b, 1H, HO—C(24)), 3.21 (m, 1H,H—C(12)), 3.15 (m, 1H, H—C(7)), 3.07 (m, 1H, H—C(3)), 2.66-2.59 (m, 12H,OCH₂CH₂CH₂S, SCH₂CH₂N), 2.18 (m, 6H, NCOCH₂CH₂), 2.15-1.06 (series ofmultiplet, 48H), 0.92 (d, 3H, J=6.5 Hz, H₃C—C(20)), 0.89 (s, 3H,H₃C—C(10)), 0.65 (s, 3H, H₃C—C(13)); ¹³C NMR (500 MHz, CDCl₃/TMS) 174.7,174.7, 174.7, 164.5, 164.5, 164.5, 164.5, 164.5, 164.5, 136.6, 136.6,136.6, 136.6, 136.6, 136.6, 85.5, 75.4, 72.4, 67.5, 67.2, 66.9, 63.3,49.9, 48.2, 45.1, 45.1, 45.1, 43.3, 41.5, 40.8, 40.8, 40.8, 39.4, 37.6,36.8, 35.3, 34.9, 34.9, 34.5, 34.5, 34.5, 34.3, 34.3, 34.3, 33.3, 33.3,33.3, 31.6, 31.0, 29.7, 29.1, 29.1, 29.0, 28.3, 28.3, 28.3, 28.1, 27.0,26.4, 26.4, 26.4, 25.7, 25.7, 25.7, 25.2, 18.5, 12.5; ESI-MS calcd. forC₆₉H₁₀₉N₆O₁₃S₃ ⁺ (M+H)⁺: 1326.71; found: 1348.09 (M+Na)⁺, 1326.17(M+H)⁺, 663.82 (M+2H)²⁺.

3α, 7α, 12α-tri-(6-Maleimido-3-thia-hexy-oxyl)-24-hydroxyl-5β-cholane(34). To a solution of free amine 32 (10 mg, 13 μmol) in 10 mL DMF wasadded N-methoxycarbonylmaleimide (20 mg, 0.13 mmol), and 1.0 mL Et₃N wasadded dropwise to the reaction mixture with stirring at roomtemperature. After stirring for 6 hours, the mixture was diluted with 20mL ethyl acetate and washed with H₂O (2×20 mL) and brine. The organiclayer was dried over anhydrous Na₂SO₄. The solvent was removed undervacuum and the residue was purified through flash chromatography withelution of ethyl acetate/hexane (5:95) to yield 34 as a light yellow oil(10 mg, 78%). ¹H NMR (500 MHz, CDCl₃/TMS) δ 6.73 (s, 6H, OCHC═CH), 3.79(d, 2H, J=2.5 Hz, H₂—C(24)), 3.74 (m, 6H, CH₂CH₂NCO), 3.68-3.57 (m, 6H,OCH₂CCH₂CH₂S), 3.57-3.42 (m, 6H, SCH₂CH₂N), 3.22 (b, 1H, HO—C(24)), 3.20(m, 1H, H—C(12)), 3.17 (m, 1H, H—C(7)), 3.05 (m, 1H, H—C(3)), 2.72 (m,6H, OCH₂CH₂CH₂S,), 2.69-2.60 (m, 6H, OCH₂CH₂CH₂S), 2.15-0.98 (series ofmultiplet, 24H), 0.92 (d, 3H, J=6.5 Hz, H₃C—C(20)), 0.88 (s, 3H,H₃C—C(10)), 0.65 (s, 3H, H₃C—C(13)); ¹³C NMR (500 MHz, CDCl₃/TMS)187.05, 176.13, 170.73, 166.02, 160.98, 157.37, 156.08, 155.85, 134.12,134.27, 134.30, 134.36, 135.06, 136.6, 104.75 ,99.57, 90.05, 60.01,57.73, 50.85, 49.9, 48.23, 37.06, 37.02, 37.10, 36.13, 29.14; 29.10,29.00. ESI-MS calcd. for C₅₁H₇₆N₃O₁₀S₃ ⁺ (M+H)⁺: 986.46; found: 986.84(M+H)⁺, 771.73 (M−215+H)⁺, 556.59. (M−2×215+H)⁺.

3α, 7α,12α-tri-[6-(2-Bromoacetylamido-3-thia-hexy-oxyl)]-24-hydroxyl-5β-cholane(35). Method A: to a solution of free amine 32 (12 mg, 16 μmol) in 10 mLDCM was added Bromoacetic anhydride (17 mg, 64 μmol). The mixture wasstirred at room temperature for 3 hour; TLC showed no more free amineremained in the reaction mixture. The solvent was removed under vacuum,the residue was purified through flash chromatography with elution ofethyl acetate/hexane (60:40) to give 35 as a pale white oil (16 mg,89%).

Method B: amine 32 (12 mg, 16 μol) was dissolved in 10 mL mixture ofMeCN/0.5 M NaHCO₃ (50:50), Bromoacetic anhydride (25 mg, 96 μmol) wasadded. The mixture was stirred for 2 hours, worked-up of the reactionmixture was in the same way as in method A to give 35 (17 mg, 96%).

¹H NMR (500 MHz, CDCl₃/TMS) of 35: δ 4.04 (m, 1H, H—C(24)), 4.00 (m, 1H,H—C(24)), 3.93 (m, 6H, OCH₂CCH₂CH₂S), 3.89 (m, 6H, CH₂CH₂NH), 3.61 (m,6H, OCH₂CH₂CH₂S), 3.32 (m, 1H, H—C(12)), 3.25 (b, 1H, HO—C(24)), 3.14(m, 7H, NCH₂CH₂S, H—C(7)), 3.08 (m, 1H, H—C(3)), 2.71 (m, 3H,OCH₂CHHCH₂S), 2.63 (m, 3H, OCH₂CHHCH₂S), 2.20-1.05 (series of multiplet,32H), 1.82 (bs, 6H, OCCH₂Br), 0.92 (d, 3H, J=6.0 Hz, H₃C—C(20)), 0.89(s, 3H, H₃C—C(10)), 0.66 (s, 3H, H₃C—C(13)); ¹³C NMR (500 MHz,CDCl₃/TMS) 80.82, 79.95, 76.15, 66.19, 65.82, 62.47, 46.52, 46.29,42.86, 42.10, 40.64, 39.87, 38.91, 38.86, 35.72, 35.45, 35.12, 34.82,32.11, 30.14, 29.93, 29.83, 28.93, 28.79, 28.61, 28.54, 28.47, 28.33,28.00, 27.65, 27.59, 27.34, 25.60, 23.32, 22.72, 22.16, 17.52, 17.13,14.47, 13.55, 11.75; ESI-MS calcd. for C₄₅H₇₉Br₃N₃O₇S₃ ⁺ (M+H)⁺:1110.26; found: 1110.52 (M+H)⁺, 555.58 (M+2H)²⁺.

General method for the ligation of thiol-containing peptides to thecholic acid-based maleimide clusters. The peptides (4.5-5.0 equivalentof maleimide) were dissolved in degassed phosphate buffer (pH6.6)/acetonitrile (1:1), maleimide 33 or 34 dissolved in acetonitrilewas added. The concentration was controlled of 2 μmol/mL approximately.The mixture was gently shaking constantly. The processing of ligationwas monitored with analytic HPLC in a proper interval of time untilstarting peptides were consumed mostly. The mixture of reaction waspurified with RP-HPLC. The collected desired fraction was lyophilized,and white cotton-like solid was obtained. The purity of the products wasconfirmed by HPLC and the identities were characterized by ESI-MS.

Trivalent peptide (36a) 83% yield; Retention time (t_(R)) 19.03 min(0.1% TFA MeCN/H₂O 0 to 90%); ESI-MS: 1889.74 (M+8H)⁸⁺, 1680.20(M+9H)⁹⁺, 1512.32 (M+10H)¹⁰⁺, 1374.92 (M+11H)¹¹⁺, 1260.44 (M+12H)¹²⁺,1163.64 (M+13H)¹³⁺, 1080.46 (M+14H)¹⁴⁺.

Trivalent peptide (36b) 91% yield; t_(R) 19.03 min (0.1% TFA MeCN/H₂O 0to 90%); ESI-MS: 155.33 (M+5H)⁵⁺, 1294.71 (M+6H)⁶⁺, 1109.88 (M+7H)⁷⁺,971.31 (M+8H)⁸⁺, 863.56 (M+9H)⁹⁺.

Trivalent peptide (37a) 90% yield; t_(R) 12.69 min (0.1% TFA MeCN/H₂O 0to 90%); ESI-MS: 1852.35 (M+2H)²⁺, 1235.16 (M+3H)³⁺, 926.63 (M+4H)⁴⁺,752.58 (M+5H)⁵⁺.

Trivalent peptide (37b) 82% yield; t_(R) 13.59 min (0.1% TFA MeCN/H₂O 0to 90%); ESI-MS: 1847.83 (M+8H)⁸⁺, 1642.74 (M+9H)⁹⁺, 1478.59 (M+10H)¹⁰⁺,1344.26 (M+11H)¹¹⁺, 1232.32 (M+12H)¹²⁺, 1137.72 (M+13H)¹³⁺, 1056.34(M+14H¹⁴⁺.

Trivalent peptide (37c) 20% yield; t_(R) 16.60 min (0.1% TFA MeCN/H₂O 0to 90%); ESI-MS: 1485.67 (M+5H)⁵⁺, 1238.18 (M+6H)⁶⁺, 1061.51 (M+7H)⁷⁺,928.94 (M+8H)⁸⁺, 825.98 (M+9H)⁹⁺.

Trivalent peptide (38). Ligation of peptide and the bromoacetylderivative. The peptide P-7C (9.7 mg, 11 μmol) was dissolved in degassed3 mL sodium borate buffer (pH 8.5)/acetonitrile (1:1) mixture.Bromoacetyl derivative 35 (2.6 mg, 2.4 μmol) dissolved in theacetonitrile was added with gentle shaking. The processing of ligationwas monitored with analytic HPLC in a proper interval of time untilstarting peptide was consumed mostly. The mixture of reaction waspurified with RP-HPLC, the collected fraction of 38 was lyophilized toprovide as a white cotton-like solid (3 mg, 35%). t_(R) 12.26 min (0.1%TFA MeCN/H₂O 0 to 90%); ESI-MS: 1792.26 (M+2H)²⁺, 1195.14 (M+3H)³⁺,896.74 (M+4H)⁴⁺, 717.68 (M+5H)⁵⁺.

Circular Dichroism (CD)

The samples of tetravalent peptide 26, trivalent peptides 36a and 37b,template 3α, 7α,12α-tri-(6-Amino-3-thia-hexy-oxyl)-24-hydroxyl-5β-cholane 32, and thesingle peptide DP178 were prepared in a concentration ranging from 2 to18 μM in 50 mM phosphate buffer (pH 7.4). The exact concentration ofeach sample was detected by UV spectrometer just prior to CDmeasurement. The measurement was run at 25° C. Blank was subtracted fromthe CD spectra, which were the average of three times scans.

The prior disclosure and examples are provided as illustration of thedisclosed invention and are not intended to limit the scope of theinvention.

REFERENCES

All cited references are herein incorporated in their entirety byreference.

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1. A maleimide cluster comprising a core carbohydrate molecule whereinthe core is selected from the group consisting of monosaccharides,oligosaccharides, and cyclic oligosaccharides and wherein at least twoor more maleimide containing groups are attached to the core, whereinthe maleimide containing groups are linked to the carbohydrate core byan alkyl cysteamine linker and optionally comprising a protein iscovalently attached to the maleimide.
 2. The maleimide cluster accordingto claim 1, wherein the core carbohydrate molecule is a monosaccharide.3. The maleimide cluster according to claim 2 wherein four or moremaleimide containing groups are attached to the core by the linker. 4.The maleimide cluster according to claim 1, wherein the core comprisescyclodextrin and wherein one or more maleimide containing groups areeach attached to the cyclodextrin.
 5. The maleimide cluster of claim 2further comprising a protein covalently attached to each of themaleimide containing groups, wherein proteins attached to the maleimidecontaining groups have the same or different amino acid sequences.
 6. Amethod of delivering a peptide drug comprising administering amultivalent peptide containing a therapeutically effective amount of thepeptide drug to a patient in need thereof, wherein the multivalentpeptide comprises peptides covalently attached to the maleimide clusterof claim
 2. 7. The method of claim 6, wherein the covalently attachedpeptides comprise the same or different amino acid sequences.
 8. Amethod of making a multivalent protein comprising contacting proteinscontaining a thiol group with the maleimide cluster according to claim 2and forming a covalent bond thereto.
 9. The method of claim 8, whereinthe covalently bonded proteins the same or different amino acidsequences.
 10. The maleimide cluster according to claim 2 comprising aprotein covalently attached to each maleimide containing groups, whereinthe protein is an HIV antigen.